Attorney Docket No.203477-768601/PCT EFFECTOR PROTEINS, COMPOSITIONS, SYSTEMS AND METHODS OF USE THEREOF FOR THE TREATMENT OF DMPK-ASSOCIATED DISEASES AND SYNDROMES CROSS-REFERENCE [1] This application claims the benefit of priority of U.S. Provisional Application No.63/514,336, filed on July 18, 2023, the entire contents of which is incorporated herein by reference. INCORPORATION BY REFERENCE OF SEQUENCE LISTING [2] The instant application contains a Sequence Listing, which has been submitted via Patent Center. The Sequence Listing titled 203477-768601_PCT_SL.xml, which was created on May 23, 2024 and is 120,978 bytes in size, is hereby incorporated by reference in its entirety. FIELD [3] The present disclosure relates generally to compositions of effector proteins and guide nucleic acids, and methods and systems of using such compositions, including detecting and editing target nucleic acids, as well as, the treatment of diseases and disorders associated with the DMPK gene. BACKGROUND [4] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins (Cas proteins), sometimes referred to as a CRISPR/Cas system, were first identified in certain bacterial species and are now understood to form part of a prokaryotic acquired immune system. CRISPR/Cas systems provide immunity in bacteria and archaea against viruses and plasmids by targeting the nucleic acids of the viruses and plasmids in a sequence-specific manner. While CRISPR/Cas proteins are involved in the acquisition, targeting and cleavage of foreign DNA or RNA, the systems may also contain a CRISPR array, which includes direct repeats flanking short spacer sequences that, in part, guide Cas proteins to their targets. SUMMARY [5] The present disclosure provides for compositions and systems comprising an effector protein, a guide nucleic acid, and uses thereof. Compositions, systems, and methods disclosed herein leverage nucleic acid modifying activities (e.g., cis cleavage activity) of these effector proteins and guide nucleic acids for the modification and detection of target nucleic acids of the DMPK gene. Accordingly, in one aspect, provided herein is a composition comprising an effector protein and a guide nucleic acid for the treatment of a disease or syndrome associated with the DMPK gene. Certain Embodiments [6] Provided herein are compositions comprising: (a) a polypeptide or a nucleic acid encoding the polypeptide; and (b) a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that is at least 90% identical to any one of the sequences 1 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT recited in TABLE 5. In some embodiments, the spacer sequence is at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences recited in TABLE 5. [7] Also provided herein are compositions comprising: (a) a polypeptide or a nucleic acid encoding the polypeptide; and (b) a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a sgRNA that is at least 85% identical to any one of the sequences recited in TABLE 8. In some embodiments, the sgRNA is at least 90%, at least 95%, at least 99%, or 100% identical to any one of the sequences recited in TABLE 8. In some embodiments, the guide nucleic acid is a sgRNA in a single nucleic acid system and the sgRNA comprises a handle sequence and a spacer sequence, and optionally wherein the handle sequence is 5’ of the spacer sequence. In some embodiments, the guide nucleic acid comprises a handle sequence that is at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 100% identical to the sequence recited in TABLE 6. [8] Also provided herein are compositions comprising: (a) a polypeptide or a nucleic acid encoding the polypeptide; and (b) a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a crRNA that is at least 85% identical to any one of the sequences recited in TABLE 7. In some embodiments, the crRNA is at least 90%, at least 95%, at least 99%, or 100% identical to any one of the sequences recited in TABLE 7. In some embodiments, the guide nucleic acid is a crRNA in a single nucleic acid system and the crRNA comprises a repeat sequence and a spacer sequence, and optionally, wherein a repeat sequence is 5’ of the spacer sequence. In some embodiments, the guide nucleic acid comprises a repeat sequence that is at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 100% identical to any one of the sequences recited in TABLE 4. In some embodiments, the guide nucleic acid comprises a repeat sequence that is at least 90% identical to SEQ ID NO: 17 or SEQ ID NO: 18. [9] In some embodiments, the composition modifies a target nucleic acid when a complex comprising the polypeptide and the guide nucleic acid hybridizes to a target sequence in a target nucleic acid. In some embodiments, the polypeptide or a nucleic acid encoding the polypeptide comprises nuclease activity. In some embodiments, the polypeptide or a nucleic acid encoding the polypeptide comprises a nuclease. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 100% identical to SEQ ID NO: 1. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 100% identical to SEQ ID NO: 2. In some embodiments, the polypeptide comprises a variant amino acid sequence of SEQ ID NO: 1, wherein the variant amino acid sequence comprises one or more amino acid alterations at one or more residues corresponding to one or more positions described in TABLE 1.1; and wherein the amino acid sequence, other than the one or more amino acid alterations has at least 90% sequence identity to the amino acid sequence referenced in SEQ ID NO: 1. 2 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [10] In some embodiments, the composition described herein includes wherein: (i) the one or more amino acid alteration is each independently a conservative or non-conservative substitution, or a combination thereof; (ii) the one or more amino acid alterations are at one or more residues corresponding to one or more positions selected from: 58, 80, 84, 105, 193, 202, 209, 210, 218, 220, 225, 246, 286, 295, 298, 306, 315, and 360, or a combination thereof, relative to SEQ ID NO: 1; (iii) the one or more amino acid alterations is each independently a substitution of an amino acid residue with a basic (positively charged) amino acid, and wherein the basic (positively charged) amino acid substitution is a substitution of an amino acid residue with a Lys (K), Arg (R), or His (H); (iv) the one or more amino acid alterations are one or more amino acid substitutions selected from: D220R, E225R, A306K, N286K, E225K, I80K, S209F, Y315M, N193K, M298L, M295W, A306K, A218K, and K58W, or a combination thereof, relative to SEQ ID NO: 1; and/or (v) the one or more amino acid alterations comprise a D220R substitution relative to SEQ ID NO: 1. [11] In some embodiments, the polypeptide comprises a variant amino acid sequence of SEQ ID NO: 2, wherein the variant amino acid sequence comprises one or more amino acid alterations at one or more residues corresponding to one or more positions described in TABLE 1.1; and wherein the amino acid sequence, other than the one or more amino acid alterations has at least 90% sequence identity to the amino acid sequence referenced in SEQ ID NO: 2. [12] In some embodiments, the composition described herein includes wherein: (i) the one or more amino acid alteration is each independently a conservative or non-conservative substitution, or a combination thereof; (ii) the one or more amino acid alterations are at one or more residues corresponding to one or more positions selected from: 2, 5, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 51, 52, 53, 54, 55, 56, 57, 59, 68, 77, 79, 84, 87, 89, 90, 92, 94, 99, 100, 101, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 147, 149, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 220, 223, 231, 240, 258, 273, 276, 281, 285, 295, 301, 304, 312, 316, 329, 334, 340, 348, 355, 357, 363, 366, 370, 392, 399, 400, 405, 406, 407, 435, 445, 471, 480, 483, 497, 501, 503, 509, 511, 512, 513, 514, 515, 516, 517, 521, 523, 526, 529, 531, 536, 540, 541, 542, 543, 544, 545, 546, 549, 568, 577, 579, 590, 591, 592, 593, 594, 595, 596, 599, 602, 603, 604, 605, 606, 607, 608, 612, 617, 620, 624, 634, 638, 639, 653, 701, and 707, or a combination thereof, relative to SEQ ID NO: 2; (iii) the one or more amino acid alterations is each independently each one or more amino acid residue alteration is independently a substitution with a basic (positively charged) amino acid, an acidic (negatively-charged) amino acid, a non-polar (hydrophobic) amino acid, or an uncharged polar amino acid, or a combination thereof; (iv) the one or more amino acid alterations is each independently a substitution of an amino acid residue with an A, N, R, K, E, S, Q, P, T, G, F or D; (v) the one or more amino acid alterations are one or more amino acid substitutions selected from: A120R, A121Q, A130R, A24R, A35R, A366V, A602R, A606R, C193R, C285V, C357L, C363V, C36R, C405L, D113R, D501K, D512R, D523K, 3 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT D549L, E100K, E101K, E109K, E109R, E119R, E258K, E31R, E33R, E34R, E42R, E44R, E529K, E536A, E595R, E68P, F14R, F202R, F312L, F39R, F445S, F509A, F53R, F701R, G111R, G122R, G136R, G13R, G179R, G25R, G276V, G32R, G497K, G55R, G56R, G577H, H110R, H208R, H20R, H604R, I2R, I126R, I127R, I17R, I191R, I203R, I240K, I471T, I59K, I603R, I653A, K118R, K128R, K135R, K15R, K184P, K184R, K189P, K200R, K206R, K29R, K348R, K37R, K38R, K392A, K99R, K281R, K407E, K435Q, K480L, K514R, K516R, K541R, K544R, K591R, K593R, K594R, K605R, K634G, K639E, K90E, K92E, K99S, L107F, L112R, L123R, L125R, L149R, L16R, L181R, L182R, L26R, L26K, L28R, L517R, L542R, L607F, L620E, M334E, M503K, M624A, N124R, N129R, N132R, N147K, N188R, N19R, N209R, N30R, N340S, N355R, N406K, N43R, N52R, N540R, N568D, N596R, P116G, P185R, P187I, P199R, P201R, P273A, P304E, P399F, P515R, P51R, P57R, P592R, P89T, P94E, P707R, Q138R, Q183R, Q195R, Q511R, Q54R, Q612R, Q79R, R18R, R22R, R329T, R41R, R531E, R546R, R617Y, S108R, S186G, S186R, S190R, S196R, S198R, S205R, S21R, S223P, S526N, S543R, S545R, S579R, S638K, S77V, T114R, T11R, T133R, T204R, T23R, T295N, T316R, T400L, T5R, T608R, T87G, V115R, V131R, V137R, V139R, V197R, V210R, V370L, V40R, V483G, V521T, V84Y, W599F, Y117R, Y134R, Y180R, Y192R, Y194R, Y207R, Y220S, Y231G, Y301L, Y513R, and Y590R, or a combination thereof, relative to SEQ ID NO: 2; and/or (vi) the one or more amino acid alteration is a L26R or L26K substitution relative to SEQ ID NO: 2. [13] In some embodiments, the polypeptide comprising a variant amino acid sequence is a variant polypeptide that generates increased indels in a target nucleic acid relative to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the variant polypeptide generates at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% more indels in a population of cells relative to a number of indels generated by a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2, as measured in a cleavage assay. In some embodiments, the polypeptide comprises at least one mutation that reduces its nuclease activity, relative to an otherwise comparable polypeptide without the mutation, as measured in a cleavage assay. In some embodiments, the variant polypeptide comprises: (i) one or more amino acid alterations, wherein the one or more amino acid alterations are one or more amino acid substitutions selected from: D237A, D418A, D418N, E335A, and E335Q, or a combination thereof, relative to SEQ ID NO: 1; or (ii) one or more amino acid alterations, wherein the one or more amino acid alterations are one or more amino acid substitutions selected from: D369A, D369N, D658A, D658N, E567A, and E567Q, or a combination thereof, relative to SEQ ID NO: 2. In some embodiments, the variant polypeptide generates decreased indels in a target nucleic acid relative to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the variant polypeptide generates about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, or about 1% less indels in a population of cells relative to a number of indels generated by a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, as measured in a cleavage assay. In some embodiments, the polypeptide is 4 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT catalytically inactive. In some embodiments, the polypeptide is fused to one or more heterologous polypeptides. [14] In some embodiments, the composition described herein include wherein: (i) the one or more heterologous polypeptides is fused to the N terminus, C terminus, or both of the polypeptide; (ii) the one or more heterologous polypeptides is directly fused to the polypeptide by an amide bond or fused by at least one linker; (iii) the one or more heterologous polypeptides comprises a nuclear localization signal (NLS); (iv) the one or more heterologous polypeptides comprises any one of the sequences of TABLE 2; and/or (v) the one or more heterologous polypeptide is an effector partner. In some embodiments, the effector partner comprises a polypeptide selected from a deaminase, a transcriptional activator, a transcriptional repressor, or a functional domain thereof. [15] In some embodiments, the polypeptide recognizes a PAM sequence comprising any one of the sequences recited in TABLE 3, and wherein optionally the PAM sequence is adjacent to a target sequence of a target nucleic acid. In some embodiments, the target nucleic acid is a human DMPK gene or comprises a mutation relative to a wild-type DMPK gene. In some embodiments, the composition modifies one or more nucleotides of a target nucleic acid. In some embodiments, the modifying comprises cleaving at least one strand of a target nucleic acid, deleting or excising one or more nucleotides of the target nucleic acid, inserting one or more nucleotides into the target nucleic acid, substituting one or more nucleotides of the target nucleic acid with one or more alternative nucleotides, or a combination thereof. In some embodiments, the composition comprises an additional engineered guide nucleic acid, or a nucleic acid encoding an additional engineered guide nucleic acid at least a portion of which hybridizes to a different target sequence of the target nucleic acid than the engineered guide nucleic acid. In some embodiments, the composition cleaves two loci of a target nucleic acid, and wherein the composition excises one or more nucleotides between the two loci of the target nucleic acid. In some embodiments, the modifying comprises deleting or excising one or more nucleotides of a target nucleic acid, wherein the one or more nucleotides are located in an untranslated region, protein coding region, an exon, an intron, a gene regulatory region, coding sequences thereof, or a combination thereof. In some embodiments, the untranslated region is the 3’ untranslated region of a target nucleic acid, and optionally wherein the 3’ untranslated region of a target nucleic acid comprises a mutation comprising expansion of a (CTG)
n repeat. In some embodiments, the expansion of the (CTG)
n repeats are: (i) greater than about (CTG)
30, about (CTG)
40, or about (CTG)
50; or (ii) about (CTG)
50 to about (CTG)
5,000. In some embodiments, a mutation is associated with a disease, and optionally wherein the disease is any one of the diseases recited in TABLE 10. [16] Provided herein are nucleic acid expression vectors encoding a guide nucleic acid comprising: (a) a spacer sequence that is at least 90% identical to any one of the sequences recited in TABLE 5, or a sgRNA that is at least 85% identical to any one of the sequences recited in TABLE 8; or (b) a spacer sequence that is at least 90% identical to any one of the sequences recited in TABLE 5, or a crRNA that is at least 85% identical to any one of the sequences recited in TABLE 7. In some embodiments, the nucleic acid expression vector is a viral vector, and optionally wherein the viral vector is an adeno associated viral 5 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT (AAV) vector. In some embodiments, the viral vector comprises a nucleotide sequence of a first promoter, wherein the first promoter drives transcription of a nucleotide sequence encoding the guide nucleic acid, and wherein the first promoter is selected from a group consisting of CMV, EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GAL1-10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, CaMV35S, SV40, CMV, 7SK, and HSV TK. In some embodiments, the nucleic acid expression vector comprises a nucleic acid sequence encoding a polypeptide that comprises an amino acid sequence that has at least 90% identity to any one of the sequences recited in TABLE 1, or a variant amino acid sequence thereof, wherein the variant amino acid sequence comprises one or more amino acid alterations, and wherein other than the one or more amino acid alterations the variant amino acid sequence has at least 80% sequence identity to any one of the sequences recited in TABLE 1. In some embodiments, the nucleic acid expression vector is a viral vector, wherein the viral vector comprises a nucleotide sequence of a second promoter, wherein the second promoter drives expression of the polypeptide, and wherein the second promoter is a ubiquitous promoter or a site-specific promoter. In some embodiments, a viral vector comprises an enhancer, wherein the enhancer is a nucleotide sequence having the effect of enhancing promoter activity, wherein the enhancer is selected from a group consisting of WPRE enhancer, CMV enhancers, the R-U5′ segment in LTR of HTLV-I, SV40 enhancer, the intron sequence between exons 2 and 3 of rabbit β-globin, and the genome region of human growth hormone. In some embodiments, a viral vector comprises a poly A signal sequence. In some embodiments, any one of the nucleic acid expression vectors described herein, wherein the nucleic acid expression vector comprises a nucleotide sequence encoding a first guide nucleic acid and a nucleotide sequence encoding a second guide nucleic acid, and wherein the first guide nucleic acid is different from the second guide nucleic acid. In some embodiments, any one of the nucleic acid expression vectors described herein, wherein a viral vector comprises a nucleotide sequence of a third promoter, wherein the third promoter drives transcription of a nucleotide sequence encoding a second guide nucleic acid, wherein the third promoter is selected from a group consisting of CMV, EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GAL1-10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, CaMV35S, SV40, CMV, 7SK, and HSV TK, and wherein a first promoter and the third promoter are different. In some embodiments, at least one nucleic acid expression vector is a lipid or a lipid nanoparticle. [17] Provided herein are pharmaceutical compositions comprising any one of the compositions described herein or any one of the nucleic acid expression vectors described herein, and a pharmaceutically acceptable excipient, carrier or diluent. [18] Also provided herein are systems comprising components for modification of a target nucleic acid, wherein the components comprise: (a) a polypeptide or a nucleic acid encoding the polypeptide; and (b) a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that is at least 90% identical to any one of the sequences recited in TABLE 5. In some embodiments, the components comprise any one of the nucleic acid expression vectors described herein. 6 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [19] Also provided herein are systems comprising components for amplification/detection of a target nucleic acid, wherein the components comprise: (a) a polypeptide or a nucleic acid encoding the polypeptide; (b) a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that is at least 90% identical to any one of the sequences recited in TABLE 5; and at least one of: (i) a detection reagent; and (ii) an amplification reagent; wherein the detection reagent is selected from: a reporter nucleic acid, a detection moiety, and an additional polypeptide, or a combination thereof; wherein the amplification reagent is selected from: a primer, a polymerase, a dNTP, and an rNTP, or a combination thereof; optionally wherein the detection reagent is operably linked to the polypeptide or the guide nucleic acid, such that a detection event occurs upon contacting the system with a target nucleic acid; and optionally wherein the amplification reagent amplifies a target nucleic acid. [20] Also provided herein are methods of modifying a target nucleic acid in a cell, the method comprising contacting the cell with the compositions described herein, the nucleic acid expression vectors described herein, the pharmaceutical compositions described herein, or the systems described herein, thereby modifying the target nucleic acid. In some embodiments, the modifying of the target nucleic acid comprises cleaving at least one strand of a target nucleic acid, deleting or excising one or more nucleotides of the target nucleic acid, inserting one or more nucleotides into the target nucleic acid, substituting one or more nucleotides of the target nucleic acid with one or more alternative nucleotides, or a combination thereof. In some embodiments, the method comprises the use of an additional engineered guide nucleic acid, or a nucleic acid encoding an additional engineered guide nucleic acid at least a portion of which hybridizes to a different target sequence of the target nucleic acid than the engineered guide nucleic acid. In some embodiments, two loci of a target nucleic acid are cleaved and the one or more nucleotides between the two loci of the target nucleic acid are excised. In some embodiments, the one or more nucleotides to be deleted or excised is an expansion of a (CTG)
n repeat in the 3’ UTR of a human DMPK gene, and optionally wherein the expansion of the (CTG)
n is greater than about (CTG)
30. [21] Provided herein are cells contacted by the compositions described herein, the nucleic acid expression vectors described herein, the pharmaceutical compositions described herein, the systems described herein, or the methods described herein. [22] Also provided herein are cells comprising a target nucleic acid modified by the compositions described herein, the nucleic acid expression vectors described herein, the pharmaceutical compositions described herein, the systems described herein, or the methods described herein. [23] Also provided herein are methods of treating a disease associated with an expansion of a (CTG)
n repeat in the 3’-untranslated region (UTR) of a human DMPK gene in a subject in need thereof, the method comprising administering to the subject the pharmaceutical compositions described herein, or the systems described herein. In some embodiments, the disease is any one of the diseases recited in TABLE 9. In some embodiments, the expansion of the (CTG)
n repeats are: (i) greater than about (CTG)
30, about (CTG)
40, or about (CTG)
50; or (ii) about (CTG)
50 to about (CTG)
5,000. 7 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT INCORPORATION BY REFERENCE [24] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS [25] FIG.1 illustrates exemplary modification activity (e.g., % indel activity) of a system comprising a CasPhi.12 L26R Variant and a single guide RNA system (as indicated in TABLE 15) for the modification/deletion of a target nucleic acid (e.g., (CTG)
n repeat region of the DMPK gene) in lipofected cells. [26] FIG. 2 illustrates exemplary modification activity (e.g., % indel activity, % modified, % full deletion) of a system comprising a CasPhi.12 L26R Variant and a dual nucleic acid system (as indicated in TABLE 16) for the modification/deletion of a target nucleic acid (e.g., (CTG)
n repeat region of the DMPK gene) in lipofected cells. DETAILED DESCRIPTION OF THE INVENTION [27] It is to be understood that both the foregoing general description and the following detailed description are exemplary, and explanatory only, and are not restrictive of the disclosure. [28] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. [29] All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. Definitions [30] Unless otherwise indicated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated or obvious from context, the following terms have the following meanings: [31] The terms, “a,” “an,” and “the,” as used herein, include plural references unless the context clearly dictates otherwise. [32] The terms, “or” and “and/or,” as used herein, include any and all combinations of one or more of the associated listed items. [33] The terms, “including,” “includes,” “included,” and other forms, are not limiting. 8 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [34] The terms, “comprise” and its grammatical equivalents, as used herein, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. [35] The term, “about,” as used herein in reference to a number or range of numbers, is understood to mean the stated number and numbers +/- 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range. [36] The terms, “% identical,” “% identity,” “percent identity,” and grammatical equivalents thereof, as used herein, in the context of an amino acid sequence or nucleotide sequence, refer to the percent of residues that are identical between respective positions of two sequences when the two sequences are aligned for maximum sequence identity. The % identity is calculated by dividing the total number of the aligned residues by the number of the residues that are identical between the respective positions of the at least two sequences and multiplying by 100. For the purposes of calculating % identity, thymine (T) may be considered the same as uracil (U). Generally, computer programs can be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci.1988 Mar;4(1):11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci U S A.1988 Apr;85(8):2444-8; Pearson, Methods Enzymol. 1990;183:63-98) and gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res.1984 Jan 11;12(1 Pt 1):387-95). [37] The terms, “% complementary”, “% complementarity”, “percent complementary”, “percent complementarity” and grammatical equivalents thereof, as used interchangeably herein, in the context of two or more nucleic acid molecules, refer to the percent of nucleotides in two nucleotide sequences in said nucleic acid molecules of equal length that can undergo cumulative base pairing at two or more individual corresponding positions in an antiparallel orientation. Accordingly, the terms include nucleic acid sequences that are not completely complementary over their entire length, which indicates that the two or more nucleic acid molecules include one or more mismatches. A “mismatch” is present at any position in the two opposed nucleotides that are not complementary. The % complementary is calculated by dividing the total number of the complementary residues by the total number of the nucleotides in one of the equal length sequences, and multiplying by 100. For the purposes of calculating % complementarity, thymine (T) may be considered the same as uracil (U). Complete or total complementarity describes nucleotide sequences in 100% of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence. “Partially complementarity” describes nucleotide sequences in which at least 20%, but less than 100%, of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence. In some instances, at least 50%, but less than 100%, of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence. In some instances, at least 70%, 80%, 90% or 95%, but less than 100%, of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence. “Noncomplementary” describes nucleotide sequences in 9 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT which less than 20% of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence. [38] The term, “percent similarity,” or “% similarity,” as used herein, in the context of an amino acid sequence, refers to a value that is calculated by dividing a similarity score by the length of the alignment. The similarity of two amino acid sequences can be calculated by using a BLOSUM62 similarity matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA., 89:10915–10919 (1992)) that is transformed so that any value ≥ 1 is replaced with +1 and any value ≤ 0 is replaced with 0. For example, an Ile (I) to Leu (L) substitution is scored at +2.0 by the BLOSUM62 similarity matrix, which in the transformed matrix is scored at +1. This transformation allows the calculation of percent similarity, rather than a similarity score. Alternately, when comparing two full protein sequences, the proteins can be aligned using pairwise MUSCLE alignment. Then, the % similarity can be scored at each residue and divided by the length of the alignment. For determining % similarity over a protein domain or motif, a multilevel consensus sequence (or PROSITE motif sequence) can be used to identify how strongly each domain or motif is conserved. In calculating the similarity of a domain or motif, the second and third levels of the multilevel sequence are treated as equivalent to the top level. Additionally, if a substitution could be treated as conservative with any of the amino acids in that position of the multilevel consensus sequence, +1 point is assigned. For example, given the multilevel consensus sequence: RLG and YCK, the test sequence QIQ would receive three points. This is because in the transformed BLOSUM62 matrix, each combination is scored as: Q-R: +1; Q-Y: +0; I-L: +1; I-C: +0; Q-G: +0; Q-K: +1. For each position, the highest score is used when calculating similarity. The % similarity can also be calculated using commercially available programs, such as the Geneious Prime software given the parameters matrix = BLOSUM62 and threshold ≥ 1. [39] The terms, “amplification,” “amplifying,” and grammatical equivalents thereof, as used herein, refer to a process by which a nucleic acid molecule is enzymatically copied to generate a plurality of nucleic acid molecules containing the same sequence as the original nucleic acid molecule or a distinguishable portion thereof. [40] The terms, “bind,” “binding,” “interact” and “interacting,” as used herein, refer to a non-covalent interaction between macromolecules (e.g., between two polypeptides, between a polypeptide and a nucleic acid; between a polypeptide/guide nucleic acid complex and a target nucleic acid; and the like). While in a state of noncovalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Non-limiting examples of non-covalent interactions are ionic bonds, hydrogen bonds, van der Waals and hydrophobic interactions. Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence-specific. [41] The term, “base editor,” as used herein, refers to a polypeptide or fusion protein comprising a base editing activity. The polypeptide with base editing activity may be referred to as an effector partner. The base editor can differ from a naturally occurring base editing enzyme. It is understood that any reference to 10 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT a base editor herein also refers to a base editing enzyme variant. The base editor is functional when the effector protein is coupled to a guide nucleic acid. The guide nucleic acid imparts sequence specific activity to the base editor. By way of non-limiting example, the effector protein may comprise a catalytically inactive effector protein (e.g., a catalytically inactive variant of an effector protein described herein). Also, by way of non-limiting example, the base editing enzyme comprises deaminase activity. [42] The term, “catalytically inactive effector protein,” as used herein, refers to an effector protein that is modified relative to a naturally-occurring effector protein to have a reduced or eliminated catalytic activity relative to that of the naturally-occurring effector protein, but retains its ability to interact with a guide nucleic acid. The catalytic activity that is reduced or eliminated is often a nuclease activity. The naturally-occurring effector protein may be a wildtype protein. In some instances, the catalytically inactive effector protein is referred to as a catalytically inactive variant of an effector protein. [43] The term, “cis cleavage,” as used herein, refers to cleavage (hydrolysis of a phosphodiester bond) of a target nucleic acid by a complex of an effector protein and a guide nucleic acid (e.g., an RNP complex), wherein at least a portion of the guide nucleic acid is hybridized to at least a portion of the target nucleic acid. In some instances, cleavage occurs within or directly adjacent to the portion of the target nucleic acid that is hybridized to the portion of the guide nucleic acid. [44] The term, “codon optimized,” as used herein, refers to a mutation of a nucleotide sequence encoding a polypeptide, such as a nucleotide sequence encoding an effector protein, to mimic the codon preferences of the intended host organism or cell while encoding the same polypeptide. Thus, the codons can be changed, but the encoded polypeptide remains unchanged. For example, if the intended target cell was a human cell, a human codon-optimized nucleotide sequence encoding an effector protein could be used. As another non-limiting example, if the intended host cell were a mouse cell, then a mouse codon- optimized nucleotide sequence encoding an effector protein could be generated. As another non-limiting example, if the intended host cell were a eukaryotic cell, then a eukaryote codon-optimized nucleotide sequence encoding an effector protein could be generated. As another non-limiting example, if the intended host cell were a prokaryotic cell, then a prokaryote codon-optimized nucleotide sequence encoding an effector protein could be generated. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.or.jp/codon. [45] The terms, “complementary” and “complementarity,” as used herein, in the context of a nucleic acid molecule or nucleotide sequence, refer to the characteristic of a polynucleotide having nucleotides that can undergo cumulative base pairing with their Watson-Crick counterparts (C with G; or A with T) in a reference nucleic acid in antiparallel orientation. For example, when every nucleotide in a polynucleotide or a specified portion thereof forms a base pair with every nucleotide in an equal length sequence of a reference nucleic acid, that polynucleotide is said to be 100% complementary to the sequence of the reference nucleic acid. In a double stranded DNA or RNA sequence, the upper (sense) strand sequence is, in general, understood as going in the direction from its 5′- to 3′-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand. 11 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT Following the same logic, the reverse sequence is understood as the sequence of the upper strand in the direction from its 3′- to its 5′-end, while the “reverse complement” sequence or the “reverse complementary” sequence is understood as the sequence of the lower strand in the direction of its 5′- to its 3′-end. Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart can be referred to as its complementary nucleotide. The complementarity of modified or artificial base pairs can be based on other types of hydrogen bonding and/or hydrophobicity of bases and/or shape complementarity between bases. [46] The term, “cleavage assay,” as used herein, refers to an assay designed to visualize, quantitate or identify cleavage of a nucleic acid. In some instances, the cleavage activity is cis cleavage activity. In some instances, the cleavage activity is trans cleavage activity. A non-limiting example of a cis cleavage assay is provided in Example 1. [47] The terms, “cleave,” “cleaving” and “cleavage,” as used herein, in the context of a nucleic acid molecule or nuclease activity of an effector protein, refer to the hydrolysis of a phosphodiester bond of a nucleic acid molecule that results in breakage of that bond. The result of this breakage can be a nick (hydrolysis of a single phosphodiester bond on one side of a double-stranded molecule), single strand break (hydrolysis of a single phosphodiester bond on a single-stranded molecule) or double strand break (hydrolysis of two phosphodiester bonds on both sides of a double-stranded molecule) depending upon whether the nucleic acid molecule is single-stranded (e.g., ssDNA or ssRNA) or double-stranded (e.g., dsDNA) and the type of nuclease activity being catalyzed by the effector protein. [48] The term, “clustered regularly interspaced short palindromic repeats (CRISPR),” as used herein, refers to a segment of DNA found in the genomes of certain prokaryotic organisms, including some bacteria and archaea, that includes repeated short sequences of nucleotides interspersed at regular intervals between unique sequences of nucleotides derived from another organism. [49] The term, “conservative substitution,” as used herein, refers to the replacement of one amino acid for another such that the replacement takes place within a family of amino acids that are related in their side chains. Conversely, the term “non-conservative substitution” as used herein refers to the replacement of one amino acid residue for another that does not have a related side chain. Genetically encoded amino acids can be divided into four families having related side chains: (1) acidic (negatively charged): Asp (D), Glu (E); (2) basic (positively charged): Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic): Cys (C), Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i) strongly hydrophobic: Ala (A), Val (V), Leu (L), Ile (I), Met (M), Phe (F); and (ii) moderately hydrophobic: Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar: Asn (N), Gln (Q), Ser (S), Thr (T). Amino acids may be related by aliphatic side chains: Gly (G), Ala (A), Val (V), Leu (L), Ile (I), Ser (S), Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic-hydroxyl; Amino acids may be related by aromatic side chains: Phe (F), Tyr (Y), Trp (W). Amino acids may be related by amide side chains: Asn (N), Gln (Q). Amino acids may be related by sulfur- containing side chains: Cys (C) and Met (M). 12 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [50] The terms, “CRISPR RNA” and “crRNA,” as used herein, refer to a type of guide nucleic acid that is RNA comprising a first sequence that is capable of hybridizing to a target sequence of a target nucleic acid and a second sequence that is capable of interacting with an effector protein either directly (by being bound by an effector protein) or indirectly (e.g., by hybridization with a second nucleic acid molecule that can be bound by an effector). The first sequence and the second sequence are directly connected to each other or by a linker. [51] The term, “detection event,” as used herein in reference to a microfluidic device, generally refers to a moment in which compositions within the detection region of a microfluidic device exhibit binding of an effector protein to a guide nucleic acid, binding of a guide nucleic acid to a target nucleic acid or target amplicon, and/or access to and cleavage of a reporter by an activated effector protein, in accordance to the assay(s) being performed. In some instances, a detection event produces a detectable product or a detectable signal. [52] The term, “detectable product,” as used herein, refers to a unit produced after the cleavage of a reporter that is capable of being discovered, identified, perceived or noticed. A detectable product can comprise a detectable label and/or moiety that emits a detectable signal. In some instances, a detectable product includes other components that are not capable of being readily discovered, identified, perceived or noticed at the same time as the detectable signal. For example, a detectable product comprises remnants of the reporter. Accordingly, in some instances, the detectable product comprises RNA and/or DNA. [53] The term, “detectable signal,” as used herein, refers to an act, event, physical quantity or impulse that can be detected using optical, fluorescent, chemiluminescent, electrochemical or other detection methods known in the art. [54] The term, “detection region,” as used herein in reference to a microfluidic device, generally refers to a structural component which comprises detection reagents that are immobilized, dried, or otherwise deposited thereto, including guide nucleic acids and/or reporters. In some instances, a detection region comprises one or more dried and/or immobilized amplification reagents including primers, polymerases, reverse transcriptase, and/or dNTPs. In some instances, a detection region comprises a single detection array, one or more lateral flow strips, a detection tray, a capture antibody, or a combination thereof. Accordingly, in some instances, a detection region comprises a plurality of microwells, detection chambers or channels, in fluid communication with amplification region(s). By way of a non-limiting example, a detection region comprises three parallel detection chambers, each coupled to a single amplification region. One of ordinary skill in the art will recognize that the relative numbers of and relationships between amplification region(s) and detection region(s) are varied depending on the assay(s) being performed. Also by way of a non-limiting example, compositions within the detection region of a microfluidic device are agitated (e.g., via a spring-loaded valve piston) to facilitate binding of an effector protein to a guide nucleic acid, binding of a guide nucleic acid to a target nucleic acid or target amplicon, and/or access to and cleavage of a reporter by an activated effector protein. 13 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [55] The term, “diseased cell,” as used herein, refers to a cell comprising pathway conditions or pathway systems that are not conducive to cell survival, tissue survival, systemic survival, or organism survival. [56] The term, “donor nucleic acid,” as used herein, refers to a nucleic acid that is (designed or intended to be) incorporated into a target nucleic acid or target sequence. [57] The term “dual nucleic acid system” as used herein refers to a system that uses a transactivated or transactivating tracrRNA-crRNA duplex complexed with one or more polypeptides described herein, wherein the complex is capable of interacting with a target nucleic acid in a sequence selective manner. [58] The term, “edited target nucleic acid,” as used herein, refers to a target nucleic acid, wherein the target nucleic acid has undergone an editing, for example, after contact with an effector protein. In some instances, the editing is an alteration in the sequence of the target nucleic acid. In some instances, the edited target nucleic acid comprises an insertion, deletion, or substitution of one or more nucleotides compared to the unedited target nucleic acid. [59] The term, “effector protein,” as used herein, refers to a protein, polypeptide, or peptide that is capable of interacting with a nucleic acid, such as a guide nucleic acid, to form a complex (e.g., a RNP complex), wherein the complex interacts with a target nucleic acid. [60] The term, “effector partner,” as used herein, refers to a protein, polypeptide or peptide that can, in combination with an effector protein and guide nucleic acid, impart some function or activity that can be used to effectuate modification(s) of a target nucleic acid described herein and/or change expression of the target nucleic acid or other nucleic acids associated with the target nucleic acid, when used in connection with compositions, systems, and methods described herein. [61] The term, “engineered modification,” as used herein, refers to a structural change of one or more nucleic acid residues of a nucleotide sequence or one or more amino acid residue of an amino acid sequence. The engineered modifications of a nucleotide sequence can include chemical modification of one or more nucleobases, or a chemical change to the phosphate backbone, a nucleotide, a nucleobase or a nucleoside. The engineered modifications can be made to an effector protein amino acid sequence or guide nucleic acid nucleotide sequence, or any sequence disclosed herein (e.g., a nucleic acid encoding an effector protein or a nucleic acid that encodes a guide nucleic acid). Methods of modifying a nucleic acid or amino acid sequence are known. One of ordinary skill in the art will appreciate that the engineered modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid, protein, composition or system is not substantially decreased. Nucleic acids provided herein can be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro-transcription, cloning, enzymatic, or chemical cleavage, etc. In some instances, the nucleic acids provided herein are not uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures can exist at various positions within the nucleic acid. [62] The term, “functional domain,” as used herein, refers to a region of one or more amino acids in a protein that is required for an activity of the protein, or the full extent of that activity, as measured in an in 14 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT vitro assay. Activities include, but are not limited to nucleic acid binding, nucleic acid editing, nucleic acid modifying, nucleic acid cleaving, protein binding. The absence of the functional domain, including mutations of the functional domain, would abolish or reduce activity. [63] The term, “functional fragment,” as used herein, refers to a fragment of a protein that retains some function relative to the entire protein. Non-limiting examples of functions are nucleic acid binding, nucleic acid editing, protein binding, nuclease activity, nickase activity, deaminase activity, demethylase activity, or acetylation activity. In some instances, a functional fragment is a recognized functional domain, e.g., a catalytic domain. [64] The term, “functional protein,” as used herein, refers to protein that retains at least some if not all activity relative to the wildtype protein. A functional protein can also include a protein having enhanced activity relative to the wildtype protein. Assays are known and available for detecting and quantifying protein activity, e.g., colorimetric and fluorescent assays. In some instances, a functional protein is a wildtype protein. In some instances, a functional protein is a functional portion of a wildtype protein. [65] The term, “fused,” as used herein, refers to at least two sequences that are connected together, such as by a covalent bond (e.g., an amide bond or a phosphodiester bond) or by a linker. The covalent bond can be formed by a conjugation (e.g., chemical conjugation or enzymatic conjugation) reaction. [66] The term, “fusion protein,” as used herein, refers to a protein comprising at least two heterologous polypeptides. In some instances, the fusion protein comprises one or more effector proteins and effector partners. In some instances, an effector protein and effector partner are not found connected to one another as a native protein or complex that occurs together in nature. [67] The term, “fusion partner,” as used herein, refers to an effector partner that is fused, or linked by a linker, to one or more effector protein. The fusion partner can impart some function or activity to the fusion protein that is not provided by the effector protein. [68] The term, “genetic disease,” as used herein, refers to a disease, disorder, condition, or syndrome associated with or caused by one or more mutations in the DNA of an organism having the genetic disease. [69] The term, “guide nucleic acid,” as used herein, refers to a nucleic acid that, when in a complex with one or more polypeptides described herein (e.g., an RNP complex) can impart sequence selectivity to the complex when the complex interacts with a target nucleic acid. In some instances, a guide nucleic acid is referred to interchangeably as a guide RNA, however it is understood that guide nucleic acids may comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), a combination thereof (e.g., RNA with a thymine base), biochemically or chemically modified nucleobases (e.g., one or more engineered modifications described herein), or a combination thereof. [70] The term, “handle sequence,” as used herein, refers to a sequence of nucleotides in a single guide RNA (sgRNA), that is: 1) capable of being non-covalently bound by an effector protein and 2) connects the portion of the sgRNA capable of being non-covalently bound by an effector protein to a nucleotide 15 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT sequence that is hybridizable to a target nucleic acid. In general, the handle sequence comprises an intermediary sequence, that is capable of being non-covalently bound by an effector protein. In some instances, the handle sequence further comprises a repeat sequence. In such instances, the intermediary sequence or a combination of the intermediary sequence and the repeat sequence is capable of being non- covalently bound by an effector protein. [71] The terms “heater”, “heating unit”, “heating element”, and “heat source”, as used herein in reference to a device, generally refers to an element that is configured to produce heat and is in thermal communication with a portion of a device. [72] The term, “heterologous,” as used herein, refers to at least two different polypeptide sequences that are not found similarly connected to one another in a native nucleic acid or protein. A protein that is heterologous to the effector protein is a protein that is not covalently linked by an amide bond to the effector protein in nature. In some instances, a protein is heterologous when the protein is not encoded by a species that encodes the effector protein. In some instances, a guide nucleic acid comprises “heterologous” sequences, which means that it includes a first sequence and a second sequence, wherein the first sequence and the second sequence are not found covalently linked by a phosphodiester bond in nature. Thus, the first sequence is considered to be heterologous with the second sequence, and, in some instances, the guide nucleic acid is referred to as a heterologous guide nucleic acid. A heterologous system comprises at least one component that is not naturally occurring together with remaining components of the heterologous system. [73] The terms, “hybridize,” “hybridizable” and grammatical equivalents thereof, refer to a nucleotide sequence that is able to noncovalently interact, i.e., form Watson-Crick base pairs and/or G/U base pairs, or anneal, to another nucleotide sequence in a sequence-specific, antiparallel, manner (i.e., a nucleotide sequence specifically interacts to a complementary nucleotide sequence) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) for both DNA and RNA. In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): guanine (G) can also base pair with uracil (U). For example, G/U base-pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, a guanine (G) can be considered complementary to both an uracil (U) and to an adenine (A). Accordingly, when a G/U base-pair can be made at a given nucleotide position, the position is not considered to be non- complementary, but is instead considered to be complementary. While hybridization typically occurs between two nucleotide sequences that are complementary, mismatches between bases are possible. It is understood that two nucleotide sequences need not be 100% complementary to be specifically hybridizable, hybridizable, partially hybridizable, or for hybridization to occur. Moreover, in some instances, a nucleotide sequence hybridizes over one or more segments such that intervening or adjacent segments are not involved 16 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.). The conditions appropriate for hybridization between two nucleotide sequences depend on the length of the sequence and the degree of complementarity, variables which are well known in the art. For hybridizations between nucleic acids with short stretches of complementarity (e.g., complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the position of mismatches may become important. Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more). Any suitable in vitro assay may be utilized to assess whether two sequences “hybridize”. One such assay is a melting point analysis where the greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Temperature, wash solution salt concentration, and other conditions may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation. Hybridization and washing conditions are well known and exemplified in Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001); and in Green, M. and Sambrook, J., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2012). [74] The term, “indel,” as used herein, refers to an insertion-deletion or indel mutation, which is a type of genetic mutation that results from the insertion and/or deletion of one or more nucleotide in a target nucleic acid. An indel can vary in length (e.g., 1 to 1,000 nucleotides in length) and be detected by any suitable method, including sequencing. [75] The term, “indel percentage,” as used herein, refers to a percentage of sequencing reads that show at least one nucleotide has been edited from the insertion and/or deletion of nucleotides regardless of the size of insertion or deletion, or number of nucleotides edited. For example, if there is at least one nucleotide deletion detected in a given target nucleic acid, it counts towards the percent indel value. As another example, if one copy of the target nucleic acid has one nucleotide deleted, and another copy of the target nucleic acid has 10 nucleotides deleted, they are counted the same. This number reflects the percentage of target nucleic acids that are edited by a given effector protein. [76] The terms, “intermediary RNA” and “intermediary sequence,” as used herein, in a context of a single nucleic acid system, refers to a nucleotide sequence in a handle sequence, wherein the nucleotide sequence is capable of, at least partially, being non-covalently bound to an effector protein to form a complex (e.g., an RNP complex). An intermediary sequence is not a transactivating nucleic acid in systems, methods, and compositions described herein. [77] The term, “in vitro,” as used herein, refers to describing something outside an organism. In some instances, an in vitro system, composition or method takes place in a container for holding laboratory reagents such that it is separated from the biological source from which a material in the container is 17 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed. The term “in vivo” is used to describe an event that takes place within an organism. The term “ex vivo” is used to describe an event that takes place in a cell that has been obtained from an organism. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. [78] The term “insertion site” as used herein refers to a location within a target nucleic acid where a donor nucleic acid is inserted. [79] The terms, “length” and “linked” as used herein, refer to a nucleic acid (polynucleotide) or polypeptide, expressed as “kilobases” (kb) or “base pairs (bp)”. Thus, a length of 1 kb refers to a length of 1000 linked nucleotides, and a length of 500 bp refers to a length of 500 linked nucleotides. Similarly, a protein having a length of 500 linked amino acids is simply described as having a length of 500 amino acids. [80] The term, “linker,” as used herein, refers to a molecule that links a first polypeptide to a second polypeptide (e.g., by an amide bond) or a first nucleic acid to a second nucleic acid (e.g., by a phosphodiester bond). [81] The terms, “mutation associated with a disease” and “mutation associated with a genetic disorder,” as used herein, refer to the co-occurrence of a mutation and the phenotype of a disease. In some instances, the mutation occurs in a gene, wherein transcription or translation products from the gene occur at a significantly abnormal level or in an abnormal form in a cell or subject harboring the mutation as compared to a non-disease control subject not having the mutation. [82] The term, “nickase,” as used herein, refers to an enzyme that possess catalytic activity for single stranded nucleic acid cleavage of a double stranded nucleic acid. [83] The term, “nickase activity,” as used herein, refers to catalytic activity that results in single stranded nucleic acid cleavage of a double stranded nucleic acid. [84] The terms, “non-naturally occurring” and “engineered,” as used herein, refer to indicate involvement of the hand of man. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid, refer to a molecule, such as but not limited to, a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid refers to a modification of that molecule (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally molecule. The terms, when referring to a composition or system described herein, refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system. By way of a non-limiting example, a composition includes an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by the hand of man. 18 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [85] The term, “NUC lobe,” as used herein, refers to the nuclease lobe which typically houses the RuvC domains. The NUC lob is connected to the REC lobe by a bridge helix. [86] The terms, “nuclease” and “endonuclease” as used herein, refer to an enzyme which possesses catalytic activity for nucleic acid cleavage. [87] The term, “nuclease activity,” as used herein, refers to catalytic activity that results in nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic acid cleavage), or deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.). [88] The term, “nucleic acid,” as used herein, refers to a polymer of nucleotides. In some instances, a nucleic acid comprises ribonucleotides, deoxyribonucleotides, combinations thereof, and modified versions of the same. In some instances, a nucleic acid is single- stranded or double-stranded, unless specified. Non- limiting examples of nucleic acids are double stranded DNA (dsDNA), single stranded (ssDNA), messenger RNA, genomic DNA, cDNA, DNA-RNA hybrids, and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Accordingly, in some instances, nucleic acids as described herein comprise one or more mutations, one or more engineered modifications, or both. [89] The term, “nucleic acid expression vector,” as used herein, refers to a plasmid that can be used to express a nucleic acid of interest. [90] The term, “nuclear localization signal (NLS),” as used herein, refers to an entity (e.g., peptide) that facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment. [91] The terms, “nucleotide(s)” and “nucleoside(s)”, as used herein, in the context of a nucleic acid molecule having multiple residues, refer to describing the sugar and base of the residue contained in the nucleic acid molecule. Similarly, a skilled artisan could understand that linked nucleotides and/or linked nucleosides, as used in the context of a nucleic acid having multiple linked residues, are interchangeable and describe linked sugars and bases of residues contained in a nucleic acid molecule. When referring to a “nucleobase(s)”, or linked nucleobase, as used in the context of a nucleic acid molecule, it can be understood as describing the base of the residue contained in the nucleic acid molecule, for example, the base of a nucleotide, nucleosides, or linked nucleotides or linked nucleosides. A person of ordinary skill in the art when referring to nucleotides, nucleosides, and/or nucleobases would also understand the differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs, such as modified uridines, do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5- methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5'-AXG where X is any modified uridine, 19 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT such as pseudouridine, NI-methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5' -CAU). [92] The term, “pharmaceutically acceptable excipient, carrier or diluent,” as used herein, refers to any substance formulated alongside the active ingredient of a pharmaceutical composition that allows the active ingredient to retain biological activity and is non-reactive with the subject's immune system. Such a substance can be included for the purpose of long-term stabilization, bulking up solid formulations that contain potent active ingredients in small amounts, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating absorption, reducing viscosity, or enhancing solubility. The selection of appropriate substance can depend upon the route of administration and the dosage form, as well as the active ingredient and other factors. Compositions having such substances can be formulated by suitable methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005). [93] The terms, “polypeptide” and “protein,” as used herein, refer to a polymeric form of amino acids. In some instances, a polypeptide includes coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Accordingly, in some instances, polypeptides as described herein comprise one or more mutations, one or more engineered modifications, or both. It is understood that when describing coding sequences of polypeptides described herein, said coding sequences do not necessarily require a codon encoding an N-terminal Methionine (M) or a Valine (V) as described for the effector proteins described herein. One skilled in the art would understand that a start codon could be replaced or substituted with a start codon that encodes for an amino acid residue sufficient for initiating translation in a host cell. In some instances, when a heterologous peptide, such as an effector partner protein, protein tag or NLS, is located at the N terminus of the effector protein, a start codon for the heterologous peptide serves as a start codon for the effector protein as well. Thus, in some instances, the natural start codon encoding an amino acid residue sufficient for initiating translation (e.g., Methionine (M) or a Valine (V)) of the effector protein is removed or absent. [94] The term, “prime editing enzyme”, as used herein, refers to a protein, polypeptide, or fragment thereof that is capable of catalyzing the editing (insertion, deletion, or base-to-base conversion) of a target nucleotide or nucleotide sequence in a nucleic acid. [95] The terms, “promoter” and “promoter sequence,” as used herein, refer to a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3’ direction) coding or non-coding sequence. A transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase, can also be found in a promoter region. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. In some instances, various promoters, including inducible promoters, are used to drive expression by the various vectors of the present disclosure. 20 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [96] The terms, “protospacer adjacent motif” and “PAM,” as used herein, refer to a nucleotide sequence found in a target nucleic acid that directs an effector protein to edit the target nucleic acid at a specific location. In some instances, a PAM is required for a complex of an effector protein and a guide nucleic acid (e.g., an RNP complex) to hybridize to and edit the target nucleic acid. In some instances, the complex does not require a PAM to edit the target nucleic acid. [97] The term “reagent mix”, “reagent master mix”, and “reagents”, as used herein, generally refers to a formulation comprising one or more chemicals that partake in a reaction that the formulation is intended for. [98] The term, “REC domain,” as used herein, refers to domain in an α-helical recognition region or lobe. In some instances, an effector protein contains at least one REC domain (e.g., REC1, REC2) which generally helps to accommodate and stabilize the guide nucleic acid and target nucleic acid hybrid. [99] The term, “recombinant,” as used herein, in the context of proteins, polypeptides, peptides and nucleic acids, refers to proteins, polypeptides, peptides and nucleic acids that are products of various combinations of cloning, restriction, and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. [100] The term, “regulatory element,” used herein, refers to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, and protein degradation signals, that provide for and/or regulate transcription of a non-coding sequence (e.g., a guide nucleic acid) or a coding sequence (e.g., effector proteins, fusion proteins) and/or regulate translation of an encoded polypeptide. [101] The term, “repeat hybridization sequence,” as used herein, in the context of a dual nucleic acid system, refers to a sequence of nucleotides of a tracrRNA that is capable of hybridizing to a repeat sequence of a guide nucleic acid. [102] The term, “repeat sequence,” as used herein, refers to a sequence of nucleotides in a guide nucleic acid that is capable of, at least partially, interacting with an effector protein. [103] The terms, “reporter,” “reporter nucleic acid,” and “reporter molecule,” as used herein, are used interchangeably and refer to a non-target nucleic acid molecule that can provide a detectable signal upon cleavage by an effector protein. Examples of detectable signals and detectable moieties that generate detectable signals are provided herein. [104] The terms, “ribonucleotide protein complex” and “RNP” as used herein, refer to a complex of one or more nucleic acids and one or more polypeptides described herein. While the term utilizes “ribonucleotides,” it is understood that the one or more nucleic acids comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), a combination thereof (e.g., RNA with a thymine base), biochemically or chemically modified nucleobases (e.g., one or more engineered modifications described herein), or combinations thereof. 21 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [105] The term, “R-Loop” as used herein, refers to a three-stranded nucleic acid structure comprising a DNA:RNA hybrid and a displaced strand of DNA. For example, an R-Loop can be formed upon hybridization of a guide nucleic acid as described herein to a target sequence of a target nucleic acid. [106] The terms, “RuvC” and “RuvC domain,” as used herein, refer to a region of an effector protein that is capable of cleaving a target nucleic acid, and in certain instances, of processing a pre-crRNA. In some instances, the RuvC domain is located near the C-terminus of the effector protein. In some instances, a single RuvC domain comprises RuvC subdomains, for example a RuvCI subdomain, a RuvCII subdomain and a RuvCIII subdomain. The term “RuvC” domain can also refer to a “RuvC-like” domain. Various RuvC-like domains are known in the art and are easily identified using online tools such as InterPro (https://www.ebi.ac.uk/interpro/). For example, in some instances, a RuvC-like domain is a domain which shares homology with a region of TnpB proteins of the IS605 and other related families of transposons. [107] The term, “sample,” as used herein, refers to something comprising a target nucleic acid. In some instances, the sample is a biological sample, such as a biological fluid or tissue sample. In some instances, the sample is an environmental sample. In some instances, the sample is a biological sample or environmental sample that is modified or manipulated. By way of non-limiting example, samples are modified or manipulated with purification techniques, heat, nucleic acid amplification, salts and buffers. [108] The terms “sample interface” and “sample input,” as used herein in reference to a microfluidic device, generally refer to a structural component capable of receiving a composition comprising a target nucleic acid as disclosed herein (e.g., a sample). In some instances, the composition comprising a target nucleic acid is a sample as defined above. In some instances, the sample is collected with a sample collector (e.g., swab, tube, etc.) before being received in a sample interface. By way of a non-limiting example, the sample is directly collected at the sample interface (e.g., without the use of a separate sample collector). In some instances, a sample interface is in fluid communication with a plurality of chambers, channels, or reservoirs of a microfluidic device. In some instances, the sample interface is fluidically connected to the plurality of chambers via lysis, preparation, amplification, or detection regions. [109] The terms, “single guide nucleic acid”, “single guide RNA” and “sgRNA,” as used herein, in the context of a single nucleic acid system, refers to a guide nucleic acid, wherein the guide nucleic acid is a single polynucleotide chain having all the required sequence for a functional complex with an effector protein (e.g., being bound by an effector protein, including in some instances activating the effector protein, and hybridizing to a target nucleic acid, without the need for a second nucleic acid molecule). For example, an sgRNA can have two or more linked guide nucleic acid components (e.g., an intermediary sequence, a repeat sequence, a spacer sequence and optionally a linker, or a handle sequence and a spacer sequence). [110] The term, “single nucleic acid system,” as used herein, refers to a system that uses a guide nucleic acid complexed with one or more polypeptides described herein, wherein the complex is capable of interacting with a target nucleic acid in a sequence specific manner, and wherein the guide nucleic acid is capable of non-covalently interacting with the one or more polypeptides described herein, and wherein the 22 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT guide nucleic acid is capable of hybridizing with a target sequence of the target nucleic acid. A single nucleic acid system lacks a duplex of a guide nucleic acid as hybridized to a second nucleic acid, wherein in such a duplex the second nucleic acid, and not the guide nucleic acid, is capable of interacting with the effector protein. In a single nucleic system, the guide nucleic acid is not transactivating or transactivated. In a single nucleic acid system, the guide nucleic acid-polypeptide complex (e.g., an RNP complex) is not transactivated or transactivating. [111] The term, “spacer sequence,” as used herein, refers to a nucleotide sequence in a guide nucleic acid that is capable of, at least partially, hybridizing to an equal length portion of a sequence (e.g., a target sequence) of a target nucleic acid. [112] The term, “subject,” as used herein, refers to an animal. In some instances, the subject is a mammal. In some instances, the subject is a human. In some instances, the subject is diagnosed or at risk for a disease. [113] The term, “sufficiently complementary,” as used herein, refers to a first nucleotide sequence that is partially complementarity to a second nucleotide sequence while still allowing the first nucleotide sequence to hybridize to the second nucleotide sequence with enough affinity to permit a biological activity to occur. Depending on the context, in some instances, a biological activity comprises the formation of a complex between two or more components described herein, such as an effector protein and a guide nucleic acid. In some instances, a biological activity comprises bringing one or more components described herein into proximity of another component, such as bringing an effector protein-guide nucleic acid complex into proximity of a target nucleic acid. In some instances, a biological activity also comprises permitting a component described herein to act on another component described herein, such as permitting an effector protein to cleave a target nucleic acid. In some instances, sequences are said to be sufficiently complementary when at least 85% of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence. [114] The term, “syndrome,” as used herein, refers to a group of symptoms which, taken together, characterize a condition. [115] The term, “target nucleic acid,” as used herein, refers to a nucleic acid that is selected as the nucleic acid for editing, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein. In some instances, target nucleic acid comprises RNA, DNA, or a combination thereof. In some instances, a target nucleic acid is single-stranded (e.g., single-stranded RNA or single-stranded DNA) or double-stranded (e.g., double-stranded DNA). [116] The term, “target sequence,” as used herein, in the context of a target nucleic acid, refers to a nucleotide sequence found within a target nucleic acid. Such a nucleotide sequence can, for example, hybridize to a respective length portion of a guide nucleic acid. [117] The terms, “trans-activating RNA”, “transactivating RNA” and “tracrRNA,” refer to a transactivating or transactivated nucleic acid in a dual nucleic acid system that is capable of hybridizing, at 23 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT least partially, to a crRNA to form a tracrRNA-crRNA duplex, and of interacting with an effector protein to form a complex (e.g., an RNP complex). [118] The terms, “transactivating”, “trans-activating”, “trans-activated”, “transactivated” and grammatical equivalents thereof, as used herein, in the context of a dual nucleic acid system refers to an outcome of the system, wherein a polypeptide is enabled to have a binding and/or nuclease activity on a target nucleic acid, by a tracrRNA or a tracrRNA-crRNA duplex. [119] The term, “trans cleavage,” as used herein, in the context of cleavage (e.g., hydrolysis of a phosphodiester bond) of one or more target nucleic acids or non-target nucleic acids, or both, by an effector protein that is complexed with a guide nucleic acid and the target nucleic acid. In some instances, Trans cleavage activity is triggered by the hybridization of a guide nucleic acid to a target nucleic acid. In some instances, the effector cleaves a target strand as well as non-target strand, wherein the target nucleic is a double stranded nucleic acid. In some instances, trans cleavage of the target nucleic acid occurs away from (e.g., not within or directly adjacent to) the portion of the target nucleic acid that is hybridized to the portion of the guide nucleic acid. [120] The term, “transcriptional activator,” as used herein, refers to a polypeptide or a fragment thereof that can activate or increase transcription of a target nucleic acid molecule. [121] The term, “transcriptional repressor,” as used herein, refers to a polypeptide or a fragment thereof that is capable of arresting, preventing, or reducing transcription of a target nucleic acid. [122] The term, “transgene,” as used herein, refers to a nucleotide sequence that is inserted into a cell for expression of said nucleotide sequence in the cell. A transgene is meant to include (1) a nucleotide sequence that is not naturally found in the cell (e.g., a heterologous nucleotide sequence); (2) a nucleotide sequence that is a mutant form of a nucleotide sequence naturally found in the cell into which it has been introduced; (3) a nucleotide sequence that serves to add additional copies of the same (e.g., exogenous or homologous) or a similar nucleotide sequence naturally occurring in the cell into which it has been introduced; or (4) a silent naturally occurring or homologous nucleotide sequence whose expression is induced in the cell into which it has been introduced. The cell in which transgene expression occurs can be a target cell, such as a host cell. [123] The term, “transposase activity,” as used herein, refers to catalytic activity that results in the transposition of a first nucleic acid into a second nucleic acid. [124] The terms, “treatment” and “treating,” as used herein, refer to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. In some instances, a therapeutic benefit refers to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, in some instances, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the 24 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or a combination thereof. For prophylactic benefit, in some instances, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease undergoes treatment, even though a diagnosis of this disease has not been made. [125] The term, “variant,” as used herein, refers to a form or version of a protein that differs from the wild-type protein. In some instances, a variant comprises a different function or activity relative to the wild- type protein. [126] The term, “viral vector,” as used herein, refers to a nucleic acid to be delivered into a host cell by a recombinantly produced virus or viral particle. Introduction [127] Disclosed herein are compositions, systems, and methods comprising at least one of: (a) a polypeptide or a nucleic acid encoding the polypeptide; and (b) a guide nucleic acid or a nucleic acid encoding the guide nucleic acid which targets DMPK. Also disclosed herein are compositions, systems, and methods comprising at least one of: (a )a polypeptide or a nucleic acid encoding the polypeptide; and (b) a guide nucleic acid or a nucleic acid encoding the guide nucleic acid which targets DMPK. Also disclosed herein are methods of using such compositions and systems for modifying or detecting a target nucleic acid(s) or one or more nucleotides of a target nucleic acid, wherein the target nucleic acid is a human DMPK gene. [128] Polypeptides described herein may bind and, optionally, cleave nucleic acids in a sequence-specific manner. Polypeptides described herein may also cleave the target nucleic acid within a target sequence or at a position adjacent to the target sequence. In some embodiments, a polypeptide is activated when it binds a certain sequence of a nucleic acid described herein, allowing the polypeptide to cleave a region of a target nucleic acid that is near, but not adjacent to the target sequence. A polypeptide may be an effector protein, such as a CRISPR-associated (Cas) protein, which may bind a guide nucleic acid that imparts activity or sequence selectivity to the polypeptide. An effector protein may also be referred to as a programmable nuclease because the nuclease activity of the protein may be directed to different target nucleic acids by way of revising the guide nucleic acid that the protein binds. [129] In some embodiments, compositions, systems, and methods comprising guide nucleic acids comprise a first region or sequence, at least a portion of which interacts with a polypeptide. In some embodiments, the first sequence comprises a sequence that is similar or identical to an intermediary nucleic acid sequence, a handle, a repeat sequence, or a combination thereof. In some embodiments, the guide nucleic acid does not comprise an intermediary nucleic acid. In some embodiments, compositions, systems, and methods comprising guide nucleic acids comprise a second sequence that is at least partially complementary to a target sequence of a target nucleic acid, and which, in some embodiments, is referred 25 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT to as a spacer sequence. In some embodiments, compositions, systems, and methods comprising effector proteins and guide nucleic acids comprise a first sequence and a second sequence, wherein the first sequence and the second sequence are heterologous to each other. In some embodiments, compositions, systems, and methods described herein further comprise an additional nucleic acid that is at least partially complementary to the first sequence as described herein. In some embodiments, the additional nucleic acid is at least partially hybridized to the 5’ end of the second region or sequence. In some embodiments, the unhybridized portion of the additional nucleic acid, at least partially interacts with the polypeptide. In some embodiments, compositions, systems, and methods described herein comprise a guide nucleic acid, wherein the guide nucleic acid comprises a crRNA or a single guide RNA (sgRNA). In some embodiments, compositions, systems, and methods described herein comprise a dual nucleic acid system. [130] In some embodiments, effector proteins disclosed herein bind and/or cleave nucleic acids, including double stranded RNA (dsRNA), single-stranded RNA (ssRNA), double stranded DNA (dsDNA), and single-stranded DNA (ssDNA). In some embodiments, polypeptides disclosed herein provide binding activity, cis cleavage activity, trans cleavage activity, nickase activity, nuclease activity, integrase activity, recombinase activity or a combination thereof. [131] The compositions, systems, and methods described herein are non-naturally occurring. In some embodiments, compositions, systems, and methods comprise an engineered guide nucleic acid (also referred to herein as a guide nucleic acid) or a use thereof. In some embodiments, compositions, systems, and methods comprise an engineered protein or a use thereof. In some embodiments, compositions, systems, and methods comprise an isolated polypeptide or a use thereof. In general, compositions, methods, and systems described herein are not found in nature. In some embodiments, compositions, methods, and systems described herein comprise at least one non-naturally occurring component. For example, in some embodiments, disclosed compositions, methods, and systems comprise a guide nucleic acid, wherein the sequence of the guide nucleic acid is different or modified from that of a naturally-occurring guide nucleic acid. [132] In some embodiments, compositions, systems, and methods comprise at least two components that do not naturally occur together. For example, in some embodiments, disclosed compositions, systems, and methods comprise a guide nucleic acid comprising a first region, at least a portion of which, interacts with a polypeptide, and a second region that is at least partially complementary to a target sequence in a target nucleic acid, wherein the first region and second region do not naturally occur together and/or are heterologous to each other. Also, by way of non-limiting example, in some embodiments, disclosed compositions, systems, and methods comprise a guide nucleic acid and an effector protein that do not naturally occur together. Likewise, by way of non-limiting example, disclosed compositions, systems, and methods comprise a ribonucleotide-protein (RNP) complex comprising an effector protein and a guide nucleic acid that do not occur together in nature. Conversely, and for clarity, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes effector proteins and guide nucleic acids from cells or organisms that have not been genetically modified by a human or machine. 26 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [133] In some embodiments, the guide nucleic acid comprises a non-natural nucleotide sequence. In some embodiments, the non-natural nucleotide sequence is a nucleotide sequence that is not found in nature. In some embodiments, the non-natural nucleotide sequence comprises a portion of a naturally-occurring sequence, wherein the portion of the naturally-occurring sequence is not present in nature absent the remainder of the naturally-occurring sequence. In some embodiments, the guide nucleic acid comprises two naturally-occurring sequences arranged in an order or proximity that is not observed in nature. In some embodiments, compositions and systems comprise a ribonucleotide complex comprising an effector protein and a guide nucleic acid that do not occur together in nature. In some embodiments, compositions and systems comprise at least two components that do not occur together in nature, wherein the at least two components comprise at least one of an effector protein, an effector partner and a guide nucleic acid. In some embodiments, guide nucleic acids comprise a first sequence and a second sequence that do not occur naturally together. For example, in some embodiments, a guide nucleic acid comprises a naturally-occurring repeat sequence and a spacer sequence that is complementary to a naturally-occurring eukaryotic sequence. In some embodiments, the guide nucleic acid comprises a repeat sequence that occurs naturally in an organism and a spacer sequence that does not occur naturally in that organism. In some embodiments, a guide nucleic acid comprises a first sequence that occurs in a first organism and a second sequence that occurs in a second organism, wherein the first organism and the second organism are different. In some embodiments, the guide nucleic acid comprises a third sequence disposed at a 3’ or 5’ end of the guide nucleic acid, or between the first and second sequences of the guide nucleic acid. In some embodiments, the guide nucleic acid comprises two heterologous sequences arranged in an order or proximity that is not observed in nature. Therefore, compositions and systems described herein are not naturally occurring. [134] In some embodiments, compositions, systems, and methods described herein comprise a polypeptide (e.g., an effector protein, an effector partner, a fusion protein, or a combination thereof) that is similar to a naturally occurring polypeptide. In some embodiments, the polypeptide lacks a portion of the naturally occurring polypeptide. In some embodiments, the polypeptide comprises a mutation relative to the naturally-occurring polypeptide, wherein the mutation is not found in nature. In some embodiments, the polypeptide also comprises at least one additional amino acid relative to the naturally-occurring polypeptide. In some embodiments, the polypeptide comprises a heterologous polypeptide. For example, in some embodiments, the polypeptide comprises an addition of a nuclear localization signal relative to the natural occurring polypeptide. In some embodiments, a nucleotide sequence encoding the polypeptide is codon optimized (e.g., for expression in a eukaryotic cell) relative to the naturally occurring sequence. Polypeptide Systems [135] Provided herein are compositions, systems and methods comprising a polypeptide or polypeptide system, wherein the polypeptide or polypeptide system described herein comprises one or more effector proteins or variants thereof, one or more effector partners or variants thereof, one or more linkers for peptides, or a combination thereof. 27 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [136] Also provided herein are compositions, systems and methods comprising a polypeptide, wherein the polypeptide comprises an effector protein, an effector partner, a fusion protein, or a combination thereof. Effector Proteins [137] Provided herein are compositions, systems, and methods comprising an effector protein or a use thereof. [138] An effector protein provided herein interacts with a guide nucleic acid to form a complex. In some embodiments, the complex interacts with a target nucleic acid. In some embodiments, an interaction between the complex and a target nucleic acid comprises one or more of: recognition of a protospacer adjacent motif (PAM) sequence within the target nucleic acid by the effector protein, hybridization of the guide nucleic acid to the target nucleic acid, modification of the target nucleic acid by the effector protein, or a combination thereof. In some embodiments, recognition of a PAM sequence within a target nucleic acid is direct the modification activity of an effector protein. In some embodiments, recognition of a PAM sequence adjacent to a target sequence of a target nucleic acid directs the modification activity of an effector protein. [139] Modification activity of an effector protein or an engineered protein described herein comprises cleavage activity, binding activity, insertion activity, or substitution activity. In some embodiments, modification activity of an effector protein results in: cleavage of at least one strand of a target nucleic acid, deletion of one or more nucleotides of a target nucleic acid, insertion of one or more nucleotides into a target nucleic acid, substitution of one or more nucleotides of a target nucleic acid with an alternative nucleotide, more than one of the foregoing, or a combination thereof. In some embodiments, modification of a target nucleic acid comprises introducing or removing epigenetic modification(s). In some embodiments, an ability of an effector protein to edit a target nucleic acid depends upon the effector protein being complexed with a guide nucleic acid, the guide nucleic acid being hybridized to a target sequence of the target nucleic acid, the distance between the target sequence and a PAM sequence, or a combination thereof. A target nucleic acid comprises a target strand and a non-target strand. Accordingly, in some embodiments, the effector protein edits a target strand and/or a non-target strand of a target nucleic acid. [140] The modification of the target nucleic acid generated by an effector protein, as a non-limiting example, results in modulation of the expression of the target nucleic acid (e.g., increasing or decreasing expression of the nucleic acid) or modulation of the activity of a translation product of the target nucleic acid (e.g., inactivation of a protein binding to an RNA molecule or hybridization). Accordingly, in some embodiments, provided herein are methods of editing a target nucleic acid using an effector protein of the present disclosure, or compositions or systems thereof. Also provided herein are methods of modulating expression of a target nucleic acid using an effector protein of the present disclosure, or compositions or systems thereof. Further provided herein are methods of modulating the activity of a translation product of a target nucleic acid using an effector protein of the present disclosure, or compositions or systems thereof. 28 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [141] In some embodiments, effector proteins disclosed herein provide nucleic acid cleavage activity, nuclease activity, or a combination thereof. In general, effector proteins described herein edit a target nucleic acid by cis cleavage activity on the target nucleic acid. In some embodiments, effector proteins disclosed herein comprise a RuvC domain capable of cleavage activity. In some embodiments, effector proteins disclosed herein cleave nucleic acids, including single stranded RNA (ssRNA), double stranded DNA (dsDNA), and single-stranded DNA (ssDNA). [142] In some embodiments, effector proteins disclosed herein provide catalytic activity (e.g., cleavage activity, nuclease activity) similar to that of a naturally-occurring effector protein, such as, for example, a naturally-occurring effector protein with reduced cleavage activity including cis cleavage activity. In some embodiments, effector proteins disclosed herein are fused to effector partners or fusion proteins wherein the effector partners or fusion proteins are capable of some function or activity not provided by an effector protein. [143] In some embodiments, an effector protein comprises a CRISPR-associated (“Cas”) protein. In some embodiments, an effector protein functions as a single protein, including a single protein that is capable of binding to a guide nucleic acid and editing a target nucleic acid. Alternatively, in some embodiments, an effector protein functions as part of a multiprotein complex, including, for example, a complex having two or more effector proteins, including two or more of the same effector proteins (e.g., dimer or multimer). In some embodiments, an effector protein, when functioning in a multiprotein complex, comprises only one functional activity (e.g., binding to a guide nucleic acid), while other effector proteins present in the multiprotein complex are capable of the other functional activity (e.g., editing a target nucleic acid). In some embodiments, an effector protein, when functioning in a multiprotein complex, comprises differing and/or complementary functional activity to other effector proteins in the multiprotein complex. Multimeric complexes, and functions thereof, are described in further detail below. In some embodiments, an effector protein comprises a modified effector protein having increased modification activity and/or increased substrate binding activity (e.g., substrate selectivity, specificity, and/or affinity). Alternatively, or in addition, an effector protein comprises a catalytically inactive effector protein having reduced modification activity or no modification activity. [144] In some embodiments, effector proteins described herein comprise one or more functional domains. Effector protein functional domains can include a protospacer adjacent motif (PAM)-interacting domain, an oligonucleotide-interacting domain, one or more recognition domains, a non-target strand interacting domain, and a RuvC domain. A PAM interacting domain can be a target strand PAM interacting domain (TPID) or a non-target strand PAM interacting domain (NTPID). In some embodiments, a PAM interacting domain, such as a TPID or a NTPID, on an effector protein describes a region of an effector protein that interacts with target nucleic acid. In some embodiments, the effector proteins comprise a RuvC domain. In some embodiments, a RuvC domain comprises with substrate binding activity, catalytic activity, or both. In some embodiments, the RuvC domain is defined by a single, contiguous sequence, or a set of RuvC subdomains that are not contiguous with respect to the primary amino acid sequence of the protein. An 29 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT effector protein of the present disclosure includes multiple RuvC subdomains, which are combined to generate a RuvC domain with substrate binding or catalytic activity. For example, in some embodiments, an effector protein comprises three RuvC subdomains (RuvC-I, RuvC-II, and RuvC-III) that are not contiguous with respect to the primary amino acid sequence of the effector protein, but form a RuvC domain once the protein is produced and folds. In some embodiments, effector proteins comprise one or more recognition domain (REC domain) with a binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex. In some embodiments, an effector protein comprises a zinc finger domain. In some embodiments, the effector protein does not comprise an HNH domain. [145] In some embodiments, an effector protein is able to recognize a variety of PAMs as described herein. In some embodiments, effector proteins described herein provide blunt or short stagger ends. In some embodiments, blunt cutting is advantageous over the staggered cutting that is provided by other effector proteins, as there is a less likely chance of spontaneous (also referred to as perfect) repair which decreases the chances of successful target nucleic acid editing and/or donor nucleic acid insertion. [146] In some embodiments, an effector protein is small, which is beneficial for nucleic acid detection or editing (for example, in some embodiments, the effector protein is less likely to adsorb to a surface or another biological species due to its small size). In some embodiments, the smaller nature of these effector proteins allow for them to be more easily packaged and delivered with higher efficiency in the context of genome editing and more readily incorporated as a reagent in an assay. In some embodiments, the length of the effector protein is at least about 100, about 200, about 300, about 400, about 500, about 600, about 700, or more linked amino acid residues. [147] In some embodiments, TABLE 1 provides illustrative amino acid sequences of effector proteins that are useful in the compositions, systems and methods described herein. In some embodiments, an effector protein, or a recombinant nucleic acid encoding an effector protein, comprises an amino acid sequence that is at least 85% identical to any one of the sequences recited in TABLE 1. In some embodiments, the recombinant nucleic acid encoding the effector protein is operably linked to a promoter, wherein the promoter is functional in an eukaryotic cell or a prokaryotic cell. In some embodiments, the promoter is any one or more of: a constitutive promoter, an inducible promoter, a cell type-specific promoter, a site-specific promoter, and a tissue-specific promoter. In some embodiments, the recombinant nucleic acid described herein wherein the promoter is functional in any one of: a plant cell, a fungal cell, an animal cell, cell of an invertebrate, a fly cell, a cell of a vertebrate, a mammalian cell, a primate cell, a non-human primate cell, and a human cell. In some embodiments, the recombinant nucleic acid is a nucleic acid expression vector as described herein. [148] In some embodiments, compositions, systems and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the amino acid sequence of the effector protein comprises at least about 200 contiguous amino acids or more of any one of the sequences recited in TABLE 1. In some embodiments, the amino acid sequence of an effector protein provided herein comprises at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 30 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400 contiguous amino acids, at least about 420 contiguous amino acids, at least about 440 contiguous amino acids, at least about 460 contiguous amino acids, at least about 480 contiguous amino acids, at least about 500 contiguous amino acids, at least about 520 contiguous amino acids, at least about 540 contiguous amino acids, at least about 560 contiguous amino acids, at least about 580 contiguous amino acids, at least about 600 contiguous amino acids, at least about 620 contiguous amino acids, at least about 640 contiguous amino acids, at least about 660 contiguous amino acids, at least about 680 contiguous amino acids, at least about 700 contiguous amino acids, at least about 720 contiguous amino acids, at least about 760 contiguous amino acids, at least about 800 contiguous amino acids, at least about 840 contiguous amino acids, at least about 880 contiguous amino acids, at least about 920 contiguous amino acids, at least about 960 contiguous amino acids, at least about 1,000 contiguous amino acids, at least about 1,100 contiguous amino acids, at least about 1,200 contiguous amino acids, at least about 1,300 contiguous amino acids, at least about 1,400 contiguous amino acids, or more of any one of the sequences of TABLE 1. [149] In some embodiments, compositions, systems and methods described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises a portion of any one of the sequences recited in TABLE 1. In some embodiments, the effector protein comprises a portion of any one of the sequences recited in TABLE 1, wherein the portion does not comprise at least the first 10 amino acids, at least the first 20 amino acids, at least the first 40 amino acids, at least the first 60 amino acids, at least the first 80 amino acids, at least the first 100 amino acids, at least the first 120 amino acids, at least the first 140 amino acids, at least the first 160 amino acids, at least the first 180 amino acids, or at least the first 200 amino acids of any one of the sequences recited in TABLE 1. In some embodiments, the effector protein comprises a portion of any one of the sequences recited in TABLE 1, wherein the portion does not comprise the last 10 amino acids, the last 20 amino acids, the last 40 amino acids, the last 60 amino acids, the last 80 amino acids, the last 100 amino acids, the last 120 amino acids, the last 140 amino acids, the last 160 amino acids, the last 180 amino acids, or the last 200 amino acids of any one of the sequences recited in TABLE 1. [150] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 65% identical to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 70% identical to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 75% identical to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 80% identical to any one of the sequences as recited in TABLE 1. In some embodiments, an 31 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT effector protein provided herein comprises an amino acid sequence that is at least 85% identical to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 90% identical to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 95% identical to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 97% identical to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 98% identical to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 99% identical to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is identical to any one of the sequences as recited in TABLE 1. [151] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% similar to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 80% similar to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 85% similar to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 90% similar to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 95% similar to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 97% similar to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 98% similar to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 99% similar to any one of the sequences as recited in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is 100% similar to any one of the sequences as recited in TABLE 1. [152] In some embodiments, compositions, systems, and methods described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more amino acid alterations relative to any one of the amino acid sequences recited in TABLE 1. In some embodiments, the effector protein comprising one or more amino acid alterations is a variant of an effector protein described herein. It is understood that any reference to an effector protein herein also refers to an effector protein variant as described herein. 32 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [153] In some embodiments, an effector protein or a nucleic acid encoding the effector protein comprises 1 amino acid alteration, 2 amino acid alterations, 3 amino acid alterations, 4 amino acid alterations, 5 amino acid alterations, 6 amino acid alterations, 7 amino acid alterations, 8 amino acid alterations, 9 amino acid alterations, 10 amino acid alterations or more relative to any one of the amino acid sequences recited in TABLE 1. In some embodiments, the one or more amino acid alterations comprises one or more amino acid substitutions, amino acid deletions, amino acid insertions, or a combination thereof. [154] In some embodiments, amino acid sequences of effector proteins described herein comprise one or more amino acid alterations relative to a reference sequence, wherein other than the one or more amino acid alterations the reference sequence is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences recited in TABLE 1. In some embodiments, amino acid sequences of effector proteins described herein comprise one or more amino acid alterations relative to a reference sequence, wherein other than the one or more amino acid alterations, the reference sequence is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% similar to any one of the amino acid sequences recited in TABLE 1. [155] In some embodiments, effector proteins provided herein are a variant of a WT effector protein, wherein the WT effector protein has an amino acid sequence of any one of the sequences recited in TABLE 1, and the effector protein comprises one or more amino acid alterations in one or more regions that interact with a substrate, such as a target nucleic acid, an engineered guide nucleic acid, or a guide nucleic acid- target nucleic acid heteroduplex. In some embodiments, effector proteins provided herein are variants of a WT effector protein, wherein the WT effector protein has an amino acid sequence of any one of the sequences recited in TABLE 1, and the effector protein comprises one or more amino acid alterations in a region of the effector protein that comprises a substrate binding activity, a catalytic activity, and/or a binding affinity for a substrate, such as a target nucleic acid, an engineered guide nucleic acid, or a guide nucleic acid-target nucleic acid heteroduplex. In some embodiments, effector proteins provided herein are a variant of a reference effector protein, wherein the reference effector protein comprises an amino acid sequence of any one of the sequences recited in TABLE 1, and the effector protein comprises one or more amino acid alterations in a RuvC domain, a REC domain, or a combination thereof. [156] The one or more amino acid alterations can be located at one or more residues corresponding to the one or more positions described in TABLE 1.1. The one or more amino acid alterations can be located at one or more residues corresponding to one or more positions in SEQ ID NO: 1. The one or more amino acid alterations can be located at one or more residues corresponding to one or more positions in SEQ ID NO: 2. As used herein, the phrase “a residue corresponding to position X in SEQ ID NO: Y” refers to a residue at a corresponding position following an alignment of two sequences. For example, the residue in SEQ ID NO: 2 corresponding to position 26 in SEQ ID NO: 1 is the residue at position 26 in SEQ ID NO: 1. 33 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [157] In some embodiments, an effector protein provided herein is a variant of a reference polypeptide, wherein the reference polypeptide has an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the effector protein has one or more amino acid alterations at one or more positions relative to SEQ ID NO: 1 or SEQ ID NO: 2, respectively. In some embodiments, an effector protein provided herein is a variant of a reference polypeptide, wherein the reference polypeptide has an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the effector protein has one or more amino acid alterations at a position described in TABLE 1.1 relative to SEQ ID NO: 1 or SEQ ID NO: 2, respectively. When describing the amino acid sequences of effector proteins described herein, a person of ordinary skill in the art understands that reference of the one or more amino acid alterations at the positions described herein (e.g., in TABLE 1.1), and the percent identity to a reference sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) describes the amino acid sequence of the effector protein itself, such that the amino acid sequence of the effector protein has the amino acid sequence of the reference sequence, but with a certain percent identity or similarity to the reference sequence while retaining the one or more amino acid alterations that the effector protein is described as having. [158] In certain embodiments, compositions, methods and systems described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein, other than the one or more amino acid alteration at one or more of the positions described in TABLE 1.1, or a combination thereof, comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences recited in TABLE 1. In certain embodiments, compositions, methods and systems described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein, other than the one or more amino acid alteration at one or more of the positions described in TABLE 1.1, or a combination thereof, comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% similar to any one of the amino acid sequences recited in TABLE 1. [159] In some embodiments, the amino acid sequence of an effector protein provided herein, other than the one or more amino acid alteration at one or more of the positions described in TABLE 1.1 or a combination thereof, comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of the amino acid sequences recited in TABLE 1. In some embodiments, the amino acid sequence of an effector protein provided herein, other than the one or more amino acid alteration at one or more of the positions described in TABLE 1.1 or a combination thereof, comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence similarity to any one of the amino acid sequences recited in TABLE 1. [160] In some embodiments, the effector protein provided herein comprises two or more simultaneous substitutions. In some embodiments, the effector protein provided herein comprises two or more simultaneous substitutions wherein each simultaneous substitution independently occurs at any one of the 34 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT one or more positions recited in TABLE 1.1 as relative to SEQ ID NO: 1. In some embodiments, the effector protein provided herein comprises two or more simultaneous substitutions wherein each simultaneous substitution independently occurs at any one of the one or more positions recited in TABLE 1.1 as relative to SEQ ID NO: 2. In some embodiments, the effector protein provided herein comprises two or more simultaneous substitutions as recited in TABLE 1.1 relative to SEQ ID NO: 2. [161] In some embodiments, each of the one or more of the amino acid alterations are at one or more residues independently corresponding to one or more positions selected from: 58, 80, 84, 105, 193, 202, 209, 210, 218, 220, 237, 418, 225, 335, 246, 286, 295, 298, 306, 315, and 360, or a combination thereof, relative to SEQ ID NO: 1. [162] In some embodiments, each of the one or more of the amino acid alterations are at one or more residues independently corresponding to one or more positions selected from: 2, 5, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 51, 52, 53, 54, 55, 56, 57, 59, 68, 77, 79, 84, 87, 89, 90, 92, 94, 99, 100, 101, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 147, 149, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 220, 223, 231, 240, 258, 273, 276, 281, 285, 295, 301, 304, 312, 316, 329, 334, 340, 348, 355, 357, 363, 366, 369, 370, 392, 399, 400, 405, 406, 407, 435, 445, 471, 480, 483, 497, 501, 503, 509, 511, 512, 513, 514, 515, 516, 517, 521, 523, 526, 529, 531, 536, 540, 541, 542, 543, 544, 545, 546, 549, 567, 568, 577, 579, 590, 591, 592, 593, 594, 595, 596, 599, 602, 603, 604, 605, 606, 607, 608, 612, 617, 620, 624, 634, 638, 639, 653, 658, 701, and 707, or a combination thereof, relative to SEQ ID NO: 2. [163] In some embodiments, the one or more amino acid alterations comprise a one or more deletions, insertions, substitutions, or a combination thereof. In some embodiments, the one or more amino acid substitutions comprise a conservative or a non-conversative substitution. As a non-limiting example, a conservative substitution relative to the L26R substitution in SEQ ID NO: 2 includes substitution of L26 for another basic (positively charged) amino acid (e.g., Lys (K), or His (H)). As a non-limiting example, a non-conservative substitution relative to the L26R substitution in SEQ ID NO: 2 includes substitution of L26 for acidic (negatively charged) amino acid (e.g., Asp (D) or Glu (E)). [164] In some embodiments, an effector protein disclosed herein comprises one or more amino acid alterations, wherein all but 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids alterations relative to the sequence in TABLE 1 are conservative amino acid substitutions. In some embodiments, an effector protein disclosed herein comprises one or more amino acid alterations, wherein all but 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids alterations relative to the sequence in TABLE 1 are non-conservative amino acid substitutions. [165] In some embodiments, each one or more amino acid alterations is independently a substitution with a basic (positively charged) amino acid, an acidic (negatively-charged) amino acid, a non-polar (hydrophobic) amino acid, or an uncharged polar amino acid, or a combination thereof. In some 35 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT embodiments, a substitution with a basic (positively charged) amino acid is a substitution of an amino acid residue with a Lys (K), Arg (R), or His (H). In some embodiments, a substitution with an acidic (negatively charged) amino acid is a substitution of an amino acid residue with an Asp (D) or Glu (E). In some embodiments, a substitution with a non-polar (hydrophobic) amino acid is a substitution of an amino acid residue with a Cys (C), Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), or Tyr (Y). In some embodiments, a substitution with an uncharged polar amino acid is a substitution of an amino acid residue with an Asn (N), Gln (Q), Ser (S), or Thr (T). In some embodiments, the one or more amino acid alterations are each a substitution of an amino acid residue with an A, N, R, K, E, S, Q, P, T, G, F or D. In some embodiments, the one or more amino acid alterations are each a substitution of an amino acid residue with an A, Q or N. In some embodiments, the one or more amino acid alterations are each a substitution of an amino acid residue with a R, K, E, S, Q, P, T, G, F or D. In some embodiments, the one or more amino acid alterations are each an alteration as described in any of TABLE 1.1, or a combination thereof, relative to SEQ ID NO: 1 or SEQ ID NO: 2 as indicated in TABLE 1.1. [166] In some embodiments, each of the one or more amino acid alterations is selected from: D220R, E225R, A306K, N286K, E225K, I80K, S209F, Y315M, N193K, M298L, M295W, A306K, A218K, K58W, D237A, D418A, D418N, E335A, and E335Q, or combinations thereof, relative to SEQ ID NO: 1. In some embodiments, the one or more amino acid alterations comprises D220R relative to SEQ ID NO: 1. [167] In some embodiments, each of the one or more amino acid alterations is selected from: A120R, A121Q, A130R, A24R, A35R, A366V, A602R, A606R, C193R, C285V, C357L, C363V, C36R, C405L, D113R, D369A, D501K, D512R, D523K, D549L, D658N, E100K, E101K, E109K, E109R, E119R, E258K, E31R, E33R, E34R, E42R, E44R, E529K, E536A, E567A, E595R, E68P, F14R, F202R, F312L, F39R, F445S, F509A, F53R, F701R, G111R, G122R, G136R, G13R, G179R, G25R, G276V, G32R, G497K, G55R, G56R, G577H, H110R, H208R, H20R, H604R, I2R, I126R, I127R, I17R, I191R, I203R, I240K, I471T, I59K, I603R, I653A, K118R, K128R, K135R, K15R, K184P, K184R, K189P, K200R, K206R, K29R, K348R, K37R, K38R, K392A, K99R, K281R, K407E, K435Q, K480L, K514R, K516R, K541R, K544R, K591R, K593R, K594R, K605R, K634G, K639E, K90E, K92E, K99S, L107F, L112R, L123R, L125R, L149R, L16R, L181R, L182R, L26R, L26K, L28R, L517R, L542R, L607F, L620E, M334E, M503K, M624A, N124R, N129R, N132R, N147K, N188R, N19R, N209R, N30R, N340S, N355R, N406K, N43R, N52R, N540R, N568D, N596R, P116G, P185R, P187I, P199R, P201R, P273A, P304E, P399F, P515R, P51R, P57R, P592R, P89T, P94E, P707R, Q138R, Q183R, Q195R, Q511R, Q54R, Q612R, Q79R, R18R, R22R, R329T, R41R, R531E, R546R, R617Y, S108R, S186G, S186R, S190R, S196R, S198R, S205R, S21R, S223P, S526N, S543R, S545R, S579R, S638K, S77V, T114R, T11R, T133R, T204R, T23R, T295N, T316R, T400L, T5R, T608R, T87G, V115R, V131R, V137R, V139R, V197R, V210R, V370L, V40R, V483G, V521T, V84Y, W599F, Y117R, Y134R, Y180R, Y192R, Y194R, Y207R, Y220S, Y231G, Y301L, Y513R, and Y590R, or a combination thereof, relative to SEQ ID NO: 36 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT 2. In some embodiments, the one or more amino acid alterations comprises recited L26R or L26K, relative to SEQ ID NO: 2. [168] A variant effector protein provided herein can comprise a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more, including up to an amino acid alteration at all of the positions identified in TABLE 1.1 relative to a wild-type effector protein (e.g., SEQ ID NO: 1 or 2). In some embodiments, an effector protein described herein has a combination of amino acid alterations comprising a first amino acid alteration at a residue corresponding to any position recited in TABLE 1.1 relative to SEQ ID NO: 1 or 2, and one or more amino acid alterations at one or more residues corresponding to any position recited in TABLE 1.1 relative to SEQ ID NO: 1 or 2 that is not at the position of the first amino acid alteration. For example, an effector protein described herein has a combination of amino acid alterations comprising a first amino acid alteration at a residue corresponding to position 26 relative to SEQ ID NO: 2, and one or more amino acid alterations at one or more residues that is not at position 26. In some embodiments, an effector protein described herein has a combination of amino acid alterations comprising two or more amino acid alterations each corresponding to any differing position as recited in TABLE 1.1 relative to SEQ ID NO: 1 or 2. [169] In some embodiments, an effector protein described herein has a combination of amino acid alterations comprising a first amino acid alteration at a residue corresponding to a position selected from: 2, 5, 26, 99, 118, 184, 198, 348, 579, 612, and 701 relative to SEQ ID NO: 2. In some embodiments, the first amino acid alteration is a substitution with a basic (positively charged) amino acid, an acidic (negatively-charged) amino acid, a non-polar (hydrophobic) amino acid, or an uncharged polar amino acid. In some embodiments, the first amino acid alteration is a substitution with an Arg (R). [170] In some embodiments, an effector protein described herein has a combination of amino acid alterations comprising a second amino acid alteration at a residue corresponding to a position selected from: 16, 26, 50, 57, 59, 70, 73, 83, 92, 94, 96, 97, 100, 109, 119, 121, 139, 150, 153, 157, 158, 186, 189, 199, 220, 223, 227, 228, 229, 230, 231, 232, 233, 234, 236, 238, 239, 241, 242, 243, 244, 245, 246, 247, 248, 250, 252, 253, 254, 255, 256, 257, 258, 259, 260, 264, 265, 266, 268, 279, 297, 361, 405, 406, 435, 471, 472, 497, 521, 568, 585, 638, 673, 674, 678, 679, 682, 684, 685, 696, 699, 703, 709, 715, and 716 relative to SEQ ID NO: 2. In some embodiments, the second amino acid alteration is a substitution with a basic (positively charged) amino acid, an acidic (negatively-charged) amino acid, a non-polar (hydrophobic) amino acid, or an uncharged polar amino acid. In some embodiments, the second amino acid alteration is a substitution with a G, R, K, E, S, Q, P, T, D, or F. [171] In some embodiments, an effector protein described herein has a combination of amino acid alterations comprising a third amino acid alteration at a residue corresponding to position 208 or 184 relative to SEQ ID NO: 2. In some embodiments, the third amino acid alteration is a substitution with a basic (positively charged) amino acid, an acidic (negatively-charged) amino acid, a non-polar (hydrophobic) amino acid, or an uncharged polar amino acid. In some embodiments, the second amino acid alteration is a substitution with a R. 37 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [172] In some embodiments, an effector protein described herein has a combination of amino acid alterations comprising a fourth amino acid alteration at a residue corresponding to position 114 relative to SEQ ID NO: 2. In some embodiments, the third amino acid alteration is a substitution with a basic (positively charged) amino acid, an acidic (negatively-charged) amino acid, a non-polar (hydrophobic) amino acid, or an uncharged polar amino acid. In some embodiments, the second amino acid alteration is a substitution with a R. [173] In some embodiments, an effector protein described herein has a combination of amino acid alterations comprising a first amino acid alteration, a second amino acid alteration, a third amino acid alteration, a fourth amino acid alteration, or a combination thereof. In some embodiments, the first amino acid alteration is at a residue corresponding to position 26 relative to SEQ ID NO: 2. In some embodiments, the first amino acid alteration is a substitution with an R. In some embodiments, the second amino acid alteration is at a residue corresponding to a position selected from: 16, 30, 38, 50, 57, 59, 70, 73, 83, 94, 96, 97, 99, 100, 108, 109, 114, 119, 149, 150, 153, 157, 158, 182, 183, 184, 198, 199, 208, 220, 223, 227, 228, 229, 230, 231, 232, 233, 234, 236, 238, 239, 241, 242, 243, 244, 245, 246, 247, 248, 250, 252, 253, 254, 255, 256, 257, 258, 259, 260, 264, 265, 266, 268, 279, 281, 297, 348, 355, 361, 405, 435, 471, 472, 497, 521, 568, 585, 638, 673, 674, 678, 679, 682, 684, 685, 696, 699, 703, 707, 709, 715, and 716 relative to SEQ ID NO: 2. In some embodiments, the second amino acid alteration is a substitution with a G, R, Q, K, E, P, T, S, D, or F. In some embodiments, the third amino acid alteration is at a residue corresponding to position 208 or 184 relative to SEQ ID NO: 2. In some embodiments, the fourth amino acid alteration at a residue corresponding to position 114 relative to SEQ ID NO: 2. In some embodiments, the third amino acid alteration and the fourth amino acid alteration is a substitution with an R. [174] In some embodiments, the first amino acid alteration is at a residue corresponding to position 184 relative to SEQ ID NO: 2. In some embodiments, the second amino acid alteration at a residue corresponding to position 183, 114, 109, 198, 208, 182, 108, or 38 relative to SEQ ID NO: 2. In some embodiments, the first amino acid alteration and the second amino acid alteration is a substitution with an R. [175] In some embodiments, the first amino acid alteration is at residue corresponding to position 5 relative to SEQ ID NO: 2. In some embodiments, the first amino acid alteration is a substitution with an R. In some embodiments, the second amino acid alteration at a residue corresponding to position 92, 121, 139, 189, 220, 223, 258, 406, 435, 471, 521, 568, or 638 relative to SEQ ID NO: 2. In some embodiments, the second amino acid alteration is a substitution with an R, Q, P, S, K, T, D, or E, or a combinations thereof. [176] In some embodiments, the first amino acid alteration is at residue corresponding to position 2 relative to SEQ ID NO: 2. In some embodiments, the second amino acid alteration at a residue corresponding to position 139 relative to SEQ ID NO: 2. In some embodiments, the first amino acid alteration and the second amino acid alteration is a substitution with an R. 38 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [177] In some embodiments, the first amino acid alteration is at residue corresponding to position 99 relative to SEQ ID NO: 2. In some embodiments, the second amino acid alteration at a residue corresponding to position 186 relative to SEQ ID NO: 2. In some embodiments, the first amino acid alteration and the second amino acid alteration is a substitution with an R. [178] In some embodiments, the first amino acid alteration is at residue corresponding to position 118 relative to SEQ ID NO: 2. In some embodiments, the first amino acid alteration is a substitution with an R. In some embodiments, the second amino acid alteration is at residue corresponding to position 92, 189, or 568 relative to SEQ ID NO: 2. In some embodiments, the second amino acid alteration is a substitution with a P, E or D, or a combination thereof. [179] In some embodiments, the first amino acid alteration is at residue corresponding to position 186 relative to SEQ ID NO: 2. In some embodiments, the first amino acid alteration is a substitution with an R. In some embodiments, the second amino acid alteration is at residue corresponding to position 258, 521, 568 relative to SEQ ID NO: 2. In some embodiments, the second amino acid alteration is a substitution with a K, T or D, or a combination thereof. [180] In some embodiments, the first amino acid alteration is at residue corresponding to position 198 relative to SEQ ID NO: 2. In some embodiments, the first amino acid alteration is a substitution with an R. In some embodiments, the second amino acid alteration is at residue corresponding to position 92, 119, 189, 220, 223, 258, 406, 471, 521, 568, or 638 relative to SEQ ID NO: 2. In some embodiments, the second amino acid alteration is a substitution with a E, S, P, K, T or D, or a combination thereof. [181] In some embodiments, the first amino acid alteration is at residue corresponding to position 348 relative to SEQ ID NO: 2. In some embodiments, the first amino acid alteration is a substitution with an R. In some embodiments, the second amino acid alteration is at residue corresponding to position 26, 92, 119, 121, 189, 220, 223, 258, 406, 435, 471, 521, or 568 relative to SEQ ID NO: 2. In some embodiments, the second amino acid alteration is a substitution with a S, Q, P, K, T, D, or E, or a combination thereof. [182] In some embodiments, the first amino acid alteration is at residue corresponding to position 579 relative to SEQ ID NO: 2. In some embodiments, the first amino acid alteration is a substitution with an R. In some embodiments, the second amino acid alteration is at residue corresponding to position 26, 92, 119, 121, 189, 220, 223, 258, 406, 435, 471, 521, 568, or 638 relative to SEQ ID NO: 2. In some embodiments, the second amino acid alteration is a substitution with a S, Q, P, K, T, D, or E, or a combination thereof. [183] In some embodiments, the first amino acid alteration is at residue corresponding to position 612 relative to SEQ ID NO: 2. In some embodiments, the first amino acid alteration is a substitution with an R. In some embodiments, the second amino acid alteration is at residue corresponding to position 26, 92, 119, 121, 189, 220, 223, 258, 406, 435, 471, 521, 568, or 638 relative to SEQ ID NO: 2. In some embodiments, the second amino acid alteration is a substitution with a S, Q, P, K, T, D, or E, or a combination thereof. 39 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [184] In some embodiments, the first amino acid alteration is at residue corresponding to position 701 relative to SEQ ID NO: 2, In some embodiments, the first amino acid alteration is a substitution with an R. In some embodiments, the second amino acid alteration is at residue corresponding to position 26, 92, 119, 121, 189, 220, 223, 258, 406, 435, 471, 521, 568, or 638 relative to SEQ ID NO: 2. In some embodiments, the second amino acid alteration is a substitution with a S, Q, P, K, T, D, or E, or a combination thereof. [185] In some embodiments, an effector protein described herein has a combination of amino acid alterations comprising a first amino acid alteration at a residue corresponding to any position recited in TABLE 1.1 relative to SEQ ID NO: 1 or 2, and one or more amino acid alterations at one or more residues corresponding to any position recited in TABLE 1.1 relative to SEQ ID NO: 1 or 2 that is not at the position of the first amino acid alteration. For example, an effector protein described herein has a combination of amino acid alterations comprising a first amino acid alteration at a residue corresponding to position 26 relative to SEQ ID NO: 2, and one or more amino acid alterations at one or more residues that is not at position 26. A person of ordinary skill in the art would readily understand when combinations of amino acid alterations are described herein, each amino acid alteration is at a different amino acid position. [186] In some embodiments, the one or more amino acid alterations are independently selected from the amino acid alterations recited in TABLE 1.1, or a combination thereof are relative to the corresponding amino acid sequence referenced in TABLE 1.1. [187] In some embodiments, the one or more amino acid alterations result in a change in activity of the effector protein relative to a naturally-occurring counterpart. For example, and as described in further detail below, the one or more amino acid alteration increases or decreases catalytic activity of the effector protein relative to a naturally-occurring counterpart. In another example, the one or more amino acid alteration increases or decreases binding activity of the effector protein relative to a naturally-occurring counterpart. In some embodiments, the one or more amino acid alterations results in a catalytically inactive effector protein variant. Catalytically inactive effect protein variants are further described herein. [188] In some embodiments, the one or more amino acid alterations result in a change in activity of the effector protein relative to a naturally-occurring counterpart. For example, and as described in further detail below, the one or more amino acid alteration increases or decreases catalytic activity of the effector protein relative to a naturally-occurring counterpart. In some embodiments, the one or more amino acid alterations results in a catalytically inactive effector protein variant. [189] In some embodiments, the one or more amino acid alterations can result in a change in activity of the effector protein relative to a naturally-occurring counterpart (a WT effector protein (e.g., SEQ ID NO: 1 or 2). For example, and as described in further detail below, the one or more amino acid alteration increases or decreases catalytic activity of the effector protein relative to a naturally-occurring counterpart (a WT effector protein (e.g., SEQ ID NO: 1 or 2)). In some embodiments, the one or more amino acid 40 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT alterations results in a catalytically inactive effector protein variant. In some embodiments, the effector proteins comprising the one or more amino acid alterations can carry out a similar enzymatic reaction as the naturally-occurring counterpart (a WT effector protein (e.g., SEQ ID NO: 1 or 2)). [190] In some embodiments, the variants of the effector protein as described herein can include amino acid alterations that provide a beneficial characteristic to effector proteins described herein, including but not limited to, increased activity (e.g., indel activity, catalytic activity, specificity or selectivity and/or affinity for a substrate, such as a target nucleic acid and/or a guide nucleic acid). In some embodiments, variants of effector proteins described herein can exhibit an activity that is at least the same or higher than the WT effector protein (e.g., SEQ ID NO: 1 or 2), that is, it has one or more activities that are the same or higher than the effector protein (e.g., SEQ ID NO: 1 or 2) without the variant at the same amino acid position(s). For example, variants can have one or more activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher over a WT effector protein (e.g., SEQ ID NO: 1 or 2). In some embodiments, activity of effector proteins described herein or variants thereof can be measured relative to a WT effector protein (e.g., SEQ ID NO: 1 or 2) in a cleavage assay, such as those described herein (see, e.g., Example 1 and 2). Effector Partners [191] Provided herein are compositions, systems, and methods comprising one or more effector partners or uses thereof. In some embodiments, the effector partner is a heterologous protein an effector protein described herein. In some embodiments, the effector partner is not an effector protein as described herein. In some embodiments, the effector partner is capable of imparting a function or activity that is not provided by an effector protein as described herein. In some embodiments, the effector partner comprises a second effector protein or a multimeric form thereof. [192] In some embodiments, an effector partner imparts a function or activity to a fusion protein comprising an effector protein that is not provided by the effector protein, including but not limited to nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, dimer forming activity (e.g., pyrimidine dimer forming activity), integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity, modification of a polypeptide associated with target nucleic acid (e.g., a histone), and/or signaling activity. 41 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [193] In some embodiments, the effector partner is fused or linked to an effector protein described herein. In some embodiments, the amino terminus of the effector partner is linked to the carboxy terminus of the effector protein directly or by a linker. In some embodiments, the carboxy terminus of the effector partner is linked to the amino terminus of the effector protein directly or by a linker. In some embodiments, the effector partner is functional when the effector protein is coupled to a guide nucleic acid. In some embodiments, the effector partner is functional when the effector protein is coupled to a target nucleic acid. In some embodiments, the guide nucleic acid imparts sequence specific activity to the effector partner. By way of non-limiting example, the effector protein comprises a catalytically inactive effector protein (e.g., a catalytically inactive variant of an effector protein described herein) when fused or linked to an effector partner. [194] In some embodiments, the effector partner directly or indirectly edits a target nucleic acid. Edits can be of a nucleobase, nucleotide, or nucleotide sequence of a target nucleic acid. In some embodiments, the effector partner interacts with additional proteins, or functional fragments thereof, to make modifications to a target nucleic acid. In other embodiments, the effector partner modifies proteins associated with a target nucleic acid. In some embodiments, an effector partner modulates transcription (e.g., inhibits transcription, increases transcription) of a target nucleic acid. In yet another example, an effector partner directly or indirectly inhibits, reduces, activates or increases expression of a target nucleic acid. Multimeric Complex Formation Modification Activity [195] In some embodiments, an effector partner inhibits the formation of a multimeric complex of an effector protein. Alternatively, the effector partner promotes the formation of a multimeric complex of the effector protein. Reverse Transcriptase (RT) Editing System [196] In some embodiments, systems and methods comprise components or uses of an RT editing system to modify a target nucleic acid. In some embodiments, RT editing is also referred to as prime editing or precise nucleobase editing. In some embodiments, an RT editing system comprises an effector protein and an effector partner comprising an RT editing enzyme. In some embodiments, the effector protein that is linked to the RT editing enzyme. In some embodiments, an RT editing enzyme comprises a polymerase. In some embodiments, an RT editing enzyme comprises a reverse transcriptase. A non-limiting example of a reverse transcriptase is an M-MLV RT enzyme and variants thereof having polymerase activity. In some embodiments, the M-MLV RT enzyme comprises at least one mutation selected from D200N, L603W, T330P, T306K, and W313F relative to wildtype M-MLV RT enzyme. In some embodiments, systems and methods comprise an RT editing enzyme, wherein the RT editing enzyme is not fused or linked to the effector protein. In some embodiments, the RT editing enzyme comprises a recruiting moiety that recruits the RT editing enzyme to the target nucleic acid. By way of non-limiting example, the RT editing enzyme comprises a peptide that binds an aptamer, wherein the aptamer is located on a guide RNA, template RNA, 42 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT or a combination thereof. Also, by way of non-limiting example, the RT editing enzyme is linked to a protein that binds to (or is bound by) the effector protein or a protein linked/fused to the effector protein. In some embodiments, an RT editing enzyme requires an RT editing guide RNA (pegRNA) to catalyze editing. In some embodiments, the pegRNA is capable of identifying a target nucleotide or target sequence in a target nucleic acid to be edited and encoding a new genetic information that replaces the target nucleotide or target sequence in the target nucleic acid. In some embodiments, an RT editing enzyme requires a pegRNA and a guide RNA, such as a single guide RNA, to catalyze the editing. In some embodiments, the RT editing system comprises a template RNA comprising a primer binding sequence that hybridizes to a primer sequence of the dsDNA molecule that is formed when target nucleic acid is cleaved, and a template sequence that is complementary to at least a portion of the target sequence of the dsDNA molecule except for at least one nucleotide. In some embodiments, the template RNA is covalently linked to a guide RNA. In some embodiments, the template RNA is not covalently linked to a guide RNA. In some embodiments, at least a portion of the template RNA hybridizes to the target nucleic acid. In some embodiments, the target nucleic acid is a dsDNA molecule. In some embodiments, at least a portion of the template RNA hybridizes to a first strand of the target nucleic acid and at least a portion of the guide RNA hybridizes to a second strand of the target nucleic acid. In some embodiments, the pegRNA comprises: a guide RNA comprising a second region that is bound by the effector protein, and a first region comprising a spacer sequence that is complementary to a target sequence of the dsDNA molecule; and a template RNA comprising a primer binding sequence that hybridizes to a primer sequence of the dsDNA molecule that is formed when target nucleic acid is cleaved, and a template sequence that is complementary to at least a portion of the target sequence of the dsDNA molecule with the exception of at least one nucleotide. In some embodiments, the at least one nucleotide is incorporated into the target nucleic acid by activity of the RT editing enzyme, thereby modifying the target nucleic acid. In some embodiments, the spacer sequence is complementary to the target sequence on a target strand of the dsDNA molecule. In some embodiments, the spacer sequence is complementary to the target sequence on a non-target strand of the dsDNA molecule. In some embodiments, the primer binding sequence hybridizes to a primer sequence on the non-target strand of the dsDNA molecule. In some embodiments, the primer binding sequence hybridizes to a primer sequence on the target strand of the dsDNA molecule. In some embodiments, the target strand is cleaved. In some embodiments, the non-target strand is cleaved. Nucleic Acid Modification Activity [197] In some embodiments, effector partners have enzymatic activity that modifies a nucleic acid, such as a target nucleic acid. In some embodiments, the target nucleic acid comprises or consists of a ssRNA, dsRNA, ssDNA, or a dsDNA. Examples of enzymatic activity that modifies the target nucleic acid include, but are not limited to: nuclease activity, which comprises the enzymatic activity of an enzyme which allows the enzyme to cleave the phosphodiester bonds between the nucleotide subunits of nucleic acids, such as that provided by a restriction enzyme, or a nuclease (e.g., FokI nuclease); methyltransferase activity such as that provided by a methyltransferase (e.g., HhaI DNA m5c-methyltransferase (M.HhaI), DNA 43 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants)); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1); DNA repair activity; DNA damage (e.g., oxygenation) activity; deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme such as rat APOBEC1); dismutase activity; alkylation activity; depurination activity; oxidation activity; pyrimidine dimer forming activity; integrase activity such as that provided by an integrase and/or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinH106Y, human immunodeficiency virus type 1 integrase (IN), Tn3 resolvase); transposase activity; recombinase activity such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase); polymerase activity; ligase activity; helicase activity; photolyase activity; and glycosylase activity. [198] In some embodiments, effector partners target a ssRNA, dsRNA, ssDNA, or a dsDNA. In some embodiments, effector partners target ssRNA. Non-limiting examples of effector partners for targeting ssRNA include, but are not limited to, splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and/or release factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and/or C to U editing enzymes); helicases; and RNA-binding proteins. [199] It is understood that an effector partner comprises an entire protein, or a fragment of the protein (e.g., a functional domain). In some embodiments, the functional domain binds or interacts with a nucleic acid, such as ssRNA, including intramolecular and/or intermolecular secondary structures thereof (e.g., hairpins, stem-loops, etc.). In some embodiments, the functional domain interacts transiently or irreversibly, directly, or indirectly. In some embodiments, a functional domain comprises a region of one or more amino acids in a protein that is required for an activity of the protein, or the full extent of that activity, as measured in an in vitro assay. Activities include but are not limited to nucleic acid binding, nucleic acid editing, nucleic acid mutating, nucleic acid modifying, nucleic acid cleaving, protein binding or combinations thereof. The absence of the functional domain, including mutations of the functional domain, would abolish or reduce activity. [200] Accordingly, in some embodiments, effector partners comprise a protein or domain thereof selected from: endonucleases (e.g., RNase III, the CRR22 DYW domain, Dicer, and PIN (PilT N-terminus); SMG5 and SMG6; domains responsible for stimulating RNA cleavage (e.g., CPSF, CstF, CFIm and CFIIm); exonucleases such as XRN-1 or Exonuclease T; deadenylases such as HNT3; protein domains responsible for nonsense mediated RNA decay (e.g., UPF1, UPF2, UPF3, UPF3b, RNP S1, Y14, DEK, REF2, and SRm160); protein domains responsible for stabilizing RNA (e.g., PABP); proteins and protein domains responsible for polyadenylation of RNA (e.g., PAP1, GLD-2, and Star- PAP); proteins and protein domains responsible for polyuridinylation of RNA (e.g., CI D1 and terminal uridylate transferase); and other suitable domains that affect nucleic acid modifications. 44 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [201] In some embodiments, effector partner comprises a chromatin-modifying enzyme. In some embodiments, the effector partner chemically modifies a target nucleic acid, for example by methylating, demethylating, or acetylating the target nucleic acid in a sequence specific or non-specific manner. Base Editing Enzymes [202] In some embodiments, effector partners edit a nucleobase of a target nucleic acid. In some embodiments, the effector partner is referred to as a base editing enzyme. In some embodiments, a base editing enzyme variant that differs from a naturally occurring base editing enzyme, but it is understood that any reference to a base editing enzyme herein also refers to a base editing enzyme variant. [203] In some embodiments, a base editor is a system comprising an effector protein and a base editing enzyme. In some embodiments, the base editor comprises a base editing enzyme and an effector protein as independent components. In some embodiments, the base editor comprises a fusion protein comprising a base editing enzyme fused or linked to an effector protein. In some embodiments, the amino terminus of the effector partner is linked to the carboxy terminus of the effector protein by the linker. In some embodiments, the carboxy terminus of the effector partner is linked to the amino terminus of the effector protein by the linker. In some embodiments, the base editor is functional when the effector protein is coupled to a guide nucleic acid. In some embodiments, the base editor is functional when the effector protein is coupled to a target nucleic acid. The guide nucleic acid imparts sequence specific activity to the base editor. By way of non-limiting example, the effector protein comprises a catalytically inactive effector protein (e.g., a catalytically inactive variant of an effector protein described herein). Also, by way of non- limiting example, the base editing enzyme comprises deaminase activity. Additional base editors are described herein. [204] In some embodiments, base editing enzymes are capable of catalyzing editing (e.g., a chemical modification) of a nucleobase of a nucleic acid molecule, such as DNA or RNA (single stranded or double stranded). In some embodiments, a base editing enzyme, and therefore a base editor, is capable of converting an existing nucleobase to a different nucleobase, such as: an adenine (A) to guanine (G); cytosine (C) to thymine (T); cytosine (C) to guanine (G); uracil (U) to cytosine (C); guanine (G) to adenine (A); hydrolytic deamination of an adenine or adenosine, or methylation of cytosine (e.g., CpG, CpA, CpT or CpC). In some embodiments, base editing enzymes edit a nucleobase on a ssDNA. In some embodiments, base editing enzymes edit a nucleobase on both strands of dsDNA. In some embodiments, base editing enzymes edit a nucleobase of an RNA. [205] In some embodiments, a base editing enzyme itself binds or does not bind to the nucleic acid molecule containing the nucleobase. In some embodiments, upon binding to its target locus in the target nucleic acid (e.g., a DNA molecule), base pairing between the guide nucleic acid and target strand leads to displacement of a small segment of ssDNA in an “R-loop”. In some embodiments, DNA bases within the R-loop are edited by the base editing enzyme having the deaminase enzyme activity. In some embodiments, base editing systems for improved efficiency in eukaryotic cells comprise a base editing enzyme, and a 45 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT catalytically inactive effector protein that generate a nick in the non-edited strand and induce repair of the non-edited strand using the edited strand as a template. [206] In some embodiments, a base editing enzyme comprises a deaminase enzyme. Exemplary deaminases are described in US20210198330, WO2021041945, WO2021050571A1, and WO2020123887, all of which are incorporated herein by reference in their entirety. Exemplary deaminase domains are described WO 2018027078 and WO2017070632, and each are hereby incorporated in its entirety by reference. Also, additional exemplary deaminase domains are described in Komor et al., Nature, 533, 420- 424 (2016); Gaudelli et al., Nature, 551, 464-471 (2017); Komor et al., Science Advances, 3:eaao4774 (2017), and Rees et al., Nat Rev Genet.2018 Dec;19(12):770-788. doi: 10.1038/s41576-018-0059-l, which are hereby incorporated by reference in their entirety. In some embodiments, the deaminase functions as a monomer. In some embodiments, the deaminase functions as heterodimer with an additional protein. In some embodiments, base editing enzymes comprise a DNA glycosylase inhibitor (e.g., an uracil glycosylase inhibitor (UGI) or uracil N-glycosylase (UNG)). In some embodiments, the effector partner is a deaminase, e.g., ADAR1/2, ADAR-2, AID, or any functional variant thereof. [207] In some embodiments, the base editor is a cytosine base editor (CBE), wherein the base editing enzyme is a cytosine base editing enzyme. In some embodiments, the cytosine base editing enzyme, and therefore CBE, converts a cytosine to a thymine. In some embodiments, a cytosine base editing enzyme accepts ssDNA as a substrate but is not capable of cleaving dsDNA, wherein the CBE comprises a catalytically inactive effector protein. In some embodiments, when bound to its cognate DNA, the catalytically inactive effector protein of the CBE performs local denaturation of the DNA duplex to generate an R-loop in which the DNA strand not paired with a guide nucleic acid exists as a disordered single- stranded bubble. In some embodiments, the catalytically inactive effector protein generated ssDNA R-loop enables the CBE to perform efficient and localized cytosine deamination in vitro. In some embodiments, deamination activity is exhibited in a window of about 4 to about 10 base pairs. In some embodiments, the catalytically inactive effector protein presents a target site to the cytosine base editing enzyme in high effective molarity, which enables the CBE to deaminate cytosines located in a variety of different sequence motifs, with differing efficacies. In some embodiments, the CBE is capable of mediating RNA-programmed deamination of target cytosines in vitro or in vivo. In some embodiments, the cytosine base editing enzyme is a cytidine deaminase. In some embodiments, the cytosine base editing enzyme is a cytosine base editing enzyme described by Koblan et al. (2018) Nature Biotechnology 36:848-846; Komor et al. (2016) Nature 533:420-424; Koblan et al. (2021) “Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning,” Nature Biotechnology; Kurt et al. (2021) Nature Biotechnology 39:41-46; Zhao et al. (2021) Nature Biotechnology 39:35-40; and Chen et al. (2021) Nature Communications 12:1384, all incorporated herein by reference. [208] In some embodiments, the effector partner comprises a uracil glycosylase inhibitor (UGI). In some embodiments, the CBE described herein comprises UGI. Base excision repair (BER) of U•G in DNA is initiated by a uracil N-glycosylase (UNG), which recognizes a U•G mismatch generated by a CBE and 46 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT cleaves the glycosidic bond between a uracil and a deoxyribose backbone of DNA. BER results in the reversion of the U•G intermediate created by the cytosine base editing enzyme back to a C•G base pair. Accordingly, in some embodiments, the UNG is inhibited by fusion of a UGI to the effector protein. In some embodiments, the UGI is a small protein from bacteriophage PBS. In some embodiments, the UGI is a DNA mimic that potently inhibits both human and bacterial UNG. In some embodiments, the UGI inhibitor is any protein or polypeptide that inhibits UNG. [209] In some embodiments, the CBE described herein mediates efficient base editing in bacterial cells and moderately efficient editing in mammalian cells, enabling conversion of a C•G base pair to a T•A base pair through a U•G intermediate. In some embodiments, the CBE is modified to increase base editing efficiency while editing more than one strand of DNA. [210] In some embodiments, the CBE described herein nicks a non-edited DNA strand. In some embodiments, the non-edited DNA strand nicked by the CBE biases cellular repair of a U•G mismatch to favor a U•A outcome, elevating base editing efficiency. [211] In some embodiments, a base editor described herein comprising one or more base editing enzymes (e.g., APOBEC1,nickase, and UGI) that efficiently edits in mammalian cells, while minimizing frequency of non-target indels. In some embodiments, base editors do not comprise a functional fragment of the base editing enzyme. In some embodiments, base editors do not comprise a function fragment of a UGI, where such a fragment is capable of excising a uracil residue from DNA by cleaving an N-glycosidic bond. [212] In some embodiments, the effector partner comprises a non-protein uracil-DNA glycosylase inhibitor (npUGI). In some embodiments, the npUGI is selected from a group of small molecule inhibitors of uracil-DNA glycosylase (UDG), or a nucleic acid inhibitor of UDG. In some embodiments, the npUGI is a small molecule derived from uracil. Examples of small molecule non-protein uracil-DNA glycosylase inhibitors, fusion proteins, and Cas-CRISPR systems comprising base editing activity are described in WO2021087246, which is incorporated by reference in its entirety. [213] In some embodiments, the base editor is a cytosine base editor, wherein the based editing enzyme is a cytosine base editing enzyme. In some embodiments, the cytosine base editing enzyme is a cytidine deaminase. In some embodiments, the base editor comprising the cytidine deaminase is generated by ancestral sequence reconstruction as described in WO2019226953, which is hereby incorporated by reference in its entirety. Non-limiting exemplary cytidine deaminases suitable for use with effector proteins described herein include: APOBEC1, APOBEC2, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, APOBEC3A, BE1 (APOBEC1-XTEN-dCas9), BE2 (APOBEC1-XTEN-dCas9- UGI), BE3 (APOBEC1-XTEN-dCas9(A840H)-UGI), BE3-Gam, saBE3, saBE4-Gam, BE4, BE4-Gam, saBE4, and saBE4-Gam as described in WO2021163587, WO2021087246, WO2021062227, and WO2020123887, which are incorporated herein by reference in their entirety. 47 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [214] In some embodiments, a base editor is a cytosine to guanine base editor (CGBE), wherein the base editing enzyme is a cytosine to guanine base editing enzyme. In some embodiments, the CGBE, converts a cytosine into a guanine. [215] In some embodiments, a base editor is an adenine base editor (ABE), wherein the base editing enzyme is an adenine base editing enzyme. In some embodiments, the adenine base editing enzyme, and therefore the ABE, converts an adenine to a guanine. In some embodiments, the adenine base editing enzyme converts an A•T base pair to a G•C base pair. In some embodiments, the adenine base editing enzyme converts a target A•T base pair to G•C in vivo or in vitro. In some embodiments, the adenine base editing enzymes provided herein reverse spontaneous cytosine deamination, which has been linked to pathogenic point mutations. In some embodiments, the adenine base editing enzymes provided herein enable correction of pathogenic SNPs (~47% of disease-associated point mutations). In some embodiments, the adenine comprises exocyclic amine that has been deaminated (e.g., resulting in altering its base pairing preferences). In some embodiments, deamination of adenosine yields inosine. In some embodiments, inosine exhibits the base-pairing preference of guanine in the context of a polymerase active site, although inosine in the third position of a tRNA anticodon is capable of pairing with A, U, or C in mRNA during translation. Non-limiting exemplary adenine base editing enzymes suitable for use with effector proteins described herein include: ABE8e, ABE8.20m, APOBEC3A, Anc APOBEC (a.k.a. AncBE4Max), and BtAPOBEC2. Non-limiting exemplary ABEs suitable for use herein include: ABE7, ABE8.1m, ABE8.2m, ABE8.3m, ABE8.4m, ABE8.5m, ABE8.6m, ABE8.7m, ABE8.8m, ABE8.9m, ABE8.10m, ABE8.11m, ABE8.12m, ABE8.13m, ABE8.14m, ABE8.15m, ABE8.16m, ABE8.17m, ABE8.18m, ABE8.19m, ABE8.20m, ABE8.21m, ABE8.22m, ABE8.23m, ABE8.24m, ABE8.1d, ABE8.2d, ABE8.3d, ABE8.4d, ABE8.5d, ABE8.6d, ABE8.7d, ABE8.8d, ABE8.9d, ABE8.10d, ABE8.11d, ABE8.12d, ABE8.13d, ABE8.14d, ABE8.15d, ABE8.16d, ABE8.17d, ABE8.18d, ABE8.19d, ABE8.20d, ABE8.21d, ABE8.22d, ABE8.23d, and ABE8.24d. In some embodiments, the adenine base editing enzyme is an adenine base editing enzyme described in Chu et al., (2021) The CRISPR Journal 4:2:169-177, incorporated herein by reference. In some embodiments, the adenine deaminase is an adenine deaminase described by Koblan et al. (2018) Nature Biotechnology 36:848-846, incorporated herein by reference. In some embodiments, the adenine base editing enzyme is an adenine base editing enzyme described by Tran et al. (2020) Nature Communications 11:4871. [216] In some embodiments, the ABE described herein is capable of targeting polyA signals, splice site acceptors, and start codons. In some embodiments, the ABE cannot create stop codons for knock-down. [217] In some embodiments, an adenine base editing enzyme is an adenosine deaminase. Non-limiting exemplary adenosine base editors suitable for use herein include ABE9. In some embodiments, the ABE comprises an engineered adenosine deaminase enzyme capable of acting on ssDNA. In some embodiments, the engineered adenosine deaminase enzyme comprises an adenosine deaminase variant that differs from a naturally occurring deaminase. Relative to the naturally occurring deaminase, in some embodiments, the adenosine deaminase variant comprises one or more amino acid alteration, including a V82S alteration, a 48 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT T166R alteration, a Y147T alteration, a Y147R alteration, a Q154S alteration, a Y123H alteration, a Q154R alteration, or a combination thereof. [218] In some embodiments, the base editor comprises an adenine deaminase (e.g., TadA). In some embodiments, the adenosine deaminase is a TadA monomer (e.g., Tad*7.10, TadA*8 or TadA*9). In some embodiments, the adenosine deaminase is a TadA*8 variant (e.g., any one of TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24 as described in WO2021163587 and WO2021050571, which are each hereby incorporated by reference in its entirety). In some embodiments, the base editor comprises TadA. [219] In some embodiments, a base editing enzyme is a deaminase dimer. In some embodiments, the ABE comprises the effector protein, the adenine base editing enzyme and the deaminase dimer. In some embodiments, the deaminase dimer comprises an adenosine deaminase. In some embodiments, the deaminase dimer comprises TadA and a suitable adenine base editing enzyme including an: ABE8e, ABE8.20m, APOBEC3A, Anc APOBEC (a.k.a. AncBE4Max), BtAPOBEC2, and variants thereof. In some embodiments, the adenine base editing enzyme is fused to amino-terminus or the carboxy-terminus of TadA. [220] In some embodiments, a base editor is an RNA base editor, wherein the base editing enzyme is an RNA base editing enzyme. In some embodiments, the RNA base editing enzyme comprises an adenosine deaminase. In some embodiments, ADAR proteins bind to RNAs and alter their sequence by changing an adenosine into an inosine. In some embodiments, RNA base editors comprise an effector protein that is activated by or binds RNA. [221] In some embodiments, base editing enzymes, and therefore base editors, are used for treating a subject having or a subject suspected of having a disease related to a gene of interest. In some embodiments, base editing enzymes, and therefore base editors, are useful for treating a disease or a disorder caused by a point mutation in a gene of interest. In some embodiments, compositions, systems, and methods described herein comprise a base editor and a guide nucleic acid, wherein the base editor comprises an effector protein and a base editing enzyme, and wherein the guide nucleic acid directs the base editor to a sequence in a target gene. Recombinases [222] In some embodiments, effector partners comprise a recombinase. In some embodiments, provided herein is a recombinase system comprising effector proteins described herein and the recombinase. In some embodiments, the effector proteins have reduced nuclease activity or no nuclease activity. In some embodiments, the recombinase is a site-specific recombinase. 49 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [223] In some embodiments, the recombinase system comprises a catalytically inactive effector protein, wherein the recombinase can be a site-specific recombinase. Such systems can be used for site-directed transgene insertion. Non-limiting examples of site-specific recombinases include a tyrosine recombinase (e.g., Cre, Flp or lambda integrase), a serine recombinase (e.g., gamma-delta resolvase, Tn3 resolvase, Sin resolvase, Gin invertase, Hin invertase, Tn5044 resolvase, IS607 transposase and integrase), or mutants or variants thereof. In some embodiments, the recombinase is a serine recombinase. Non-limiting examples of serine recombinases include gamma-delta resolvase, Tn3 resolvase, Sin resolvase, Gin invertase, Hin invertase, Tn5044 resolvase, IS607 transposase, and IS607 integrase. In some embodiments, the site- specific recombinase is an integrase. Non-limiting examples of integrases include:Bxb1, wBeta, BL3, phiR4, A118, TG1, MR11, phi370, SPBc, TP901-1, phiRV, FC1, K38, phiBT1, and phiC31. Further discussion and examples of suitable recombinase effector partners are described in US 10,975,392, which is incorporated herein by reference in its entirety. In some embodiments, the fusion protein comprises a linker that links the recombinase to the Cas-CRISPR domain of the effector protein. In some embodiments, the linker is The-Ser. Linkers for peptides [224] In some embodiments, a linker comprises a bond or molecule that links a first polypeptide to a second polypeptide. Accordingly, in some embodiments, effector proteins, effector partners, or a combination thereof are connected by linkers. In some embodiments, the linker comprises or consists of a covalent bond. In some embodiments, the linker comprises or consists of a chemical group. In some embodiments, the linker comprises an amino acid. In some embodiments, a peptide linker comprises at least two amino acids linked by an amide bond. In general, the linker connects a terminus of the effector protein to a terminus of the effector partner. In some embodiments, carboxy terminus of the effector protein is linked to the amino terminus of the fusion effector. In some embodiments, carboxy terminus of the effector partner is linked to the amino terminus of the effector protein. In some embodiments, the effector protein and the effector partner are directly linked by a covalent bond. [225] In some embodiments, linkers comprise one or more amino acids. In some embodiments, linker is a protein. In some embodiments, a terminus of the effector protein is linked to a terminus of the effector partner through an amide bond. In some embodiments, a terminus of the effector protein is linked to a terminus of the effector partner through a peptide bond. In some embodiments, linkers comprise an amino acid. In some embodiments, linkers comprise a peptide. In some embodiments, an effector protein is coupled to an effector partner by a linker protein. In some embodiments, the linker comprises any of a variety of amino acid sequences. In some embodiments, the linker comprises a region of rigidity (e.g., beta sheet, alpha helix), a region of flexibility, or a combination thereof. In some embodiments, the linker comprises small amino acids, such as glycine and alanine, that impart high degrees of flexibility. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any desired element comprises linkers that are all or partially flexible, such that the linker comprises a flexible linker as well as one or more portions that confer less flexible structure. Suitable linkers include proteins of 4 linked amino 50 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT acids to 40 linked amino acids in length, or between 4 linked amino acids and 25 linked amino acids in length. In some embodiments, linked amino acids described herein comprise at least two amino acids linked by an amide bond. [226] In some embodiments, linkers are produced by using synthetic, linker-encoding oligonucleotides to couple proteins, or are encoded by a nucleic acid sequence encoding a fusion protein (e.g., an effector protein coupled to an effector partner). In some embodiments, the linker is from 1 to 300, from 1 to 250, from 1 to 200, from 1 to 150, from 1 to 100, from 1 to 50, from 1 to 25, from 1 to 10, from 10 to 300, from 10 to 250, from 10 to 200, from 10 to 150, from 10 to 100, from 10 to 50, from 10 to 25, from 25 to 300, from 25 to 250, from 25 to 200, from 25 to 150, from 25 to 100, from 25 to 50, from 50 to 300, from 50 to 250, from 50 to 200, from 50 to 150, from 50 to 100, from 100 to 300, from 100 to 250, from 100 to 200, from 100 to 150, from 150 to 300, from 150 to 250, from 150 to 200, from 200 to 300, from 200 to 250, or from 250 to 300 amino acids in length. In some embodiments, the linker is from 1 to 100 amino acids in length. In some embodiments, the linker is more 100 amino acids in length. In some embodiments, the linker is from 10 to 27 amino acids in length. In some embodiments, linker proteins include glycine polymers (G)
n, glycine-serine polymers (including, for example, (GS)
n, GSGGS
n (SEQ ID NO: 99), GGSGGS
n (SEQ ID NO: 100), and GGGS
n (SEQ ID NO: 101), where n is an integer of at least one), glycine-alanine polymers, and alanine-serine polymers. In some embodiments, linkers comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 102), GGSGG (SEQ ID NO: 103), GSGSG (SEQ ID NO: 104), GSGGG (SEQ ID NO: 105), GGGSG (SEQ ID NO: 106), and GSSSG (SEQ ID NO: 107). In some embodiments, the linker comprises one or more repeats a tri-peptide GGS. In some embodiments, the linker is an XTEN linker. In some embodiments, the XTEN linker is an XTEN80 linker. In some embodiments, the XTEN linker is an XTEN20 linker. In some embodiments, the XTEN20 linker has an amino acid sequence of GSGGSPAGSPTSTEEGTSESATPGSG [SEQ ID NO: 108]. [227] In some embodiments, linkers do not comprise an amino acid. In some embodiments, linkers do not comprise a peptide. In some embodiments, linkers comprise a nucleotide, a polynucleotide, a polymer, or a lipid. In some embodiments, a linker comprises a polyethylene glycol (PEG), polypropylene glycol (PPG), co-poly(ethylene/propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinylpyrrolidones, polyvinyl ethyl ether, polyacrylamide, polyacrylate, polycyanoacrylates, lipid polymers, chitins, hyaluronic acid, heparin, or an alkyl linker. [228] In some embodiments, a linker is recognized and cleaved by a protein. In some embodiments, a linker comprises a recognition sequence. In some embodiments, the recognition sequence is recognized and cleaved by the protein. In some embodiments, a guide nucleic acid comprises an aptamer. In some embodiments, the aptamer serves a similar function as a linker, bringing an effector protein and an effector partner protein into proximity. In some embodiments, the aptamer functionally connects two proteins (e.g., effector protein, effector partner) by interacting non-covalently with both, thereby bringing both proteins into proximity of the guide nucleic acid. In some embodiments, the first protein and/or the second protein comprise or is covalently linked to an aptamer binding moiety. In some embodiments, the aptamer is a short 51 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT single stranded DNA (ssDNA) or RNA (ssRNA) molecule capable of being bound be the aptamer binding moiety. In some embodiments, the aptamer is a molecule that is capable of mimicking antibody binding activity. In some embodiments, the aptamer is classified as a chemical antibody. In some instances, the aptamer described herein refers to artificial oligonucleotides that bind one or more specific molecules. In some embodiments, aptamers exhibit a range of affinities (K
D in the pM to μM range) with little or no off- target binding. Engineered Proteins [229] In some embodiments, proteins (e.g., effector protein or effector partner) described herein have been modified (also referred to as an engineered protein). In some embodiments, a modification of the proteins comprises addition of one or more amino acids, deletion of one or more amino acids, substitution of one or more amino acids, or a combination thereof. In some embodiments, the proteins disclosed herein are engineered proteins. Unless otherwise indicated, reference to the proteins throughout the present disclosure include engineered proteins thereof. [230] In some embodiments, proteins (e.g., effector protein or effector partner) described herein can be modified with the addition of one or more heterologous peptides. In some embodiments, the protein modified with the addition of one or more heterologous peptides is referred to herein as a fusion protein. Such fusion proteins are described herein and throughout. [231] In some embodiments, a heterologous peptide comprises a subcellular localization signal. In some embodiments, a subcellular localization signal can be a nuclear localization signal (NLS). In some embodiments, the NLS facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment. TABLE 2 lists exemplary NLS sequences. In some embodiments, the subcellular localization signal is a nuclear export signal (NES), a sequence to keep the protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, or an ER retention signal. In some embodiments, the protein described herein is not modified with a subcellular localization signal so that the protein is not targeted to the nucleus, which can be advantageous depending on the circumstance (e.g., when the target nucleic acid is an RNA that is present in the cytosol). [232] In some embodiments, a heterologous peptide comprises a chloroplast transit peptide (CTP), also referred to as a chloroplast localization signal or a plastid transit peptide, which targets the protein to a chloroplast. Chromosomal transgenes from bacterial sources require a sequence encoding a CTP sequence fused to a sequence encoding an expressed protein (e.g., effector protein, effector partner) if the expressed protein is to be compartmentalized in the plant plastid (e.g., chloroplast). In some embodiments, the CTP is removed in a processing step during translocation into the plastid. Accordingly, localization of the protein to a chloroplast is often accomplished by means of operably linking a polynucleotide sequence encoding a CTP sequence to the 5' region of a polynucleotide encoding the exogenous protein. 52 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [233] In some embodiments, the heterologous peptide is an endosomal escape peptide (EEP). An EEP is an agent that quickly disrupts the endosome in order to minimize the amount of time that a delivered molecule, such protein, spends in the endosome-like environment, and to avoid getting trapped in the endosomal vesicles and degraded in the lysosomal compartment. An exemplary EEP is recited in TABLE 2. [234] In some embodiments, the heterologous peptide is a cell penetrating peptide (CPP), also known as a Protein Transduction Domain (PTD). A CPP or PTD is a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. [235] Further suitable heterologous peptides include, but are not limited to, proteins (or fragments/domains thereof) that are boundary elements (e.g., CTCF), proteins and fragments thereof that provide periphery recruitment (e.g., Lamin A, Lamin B, etc.), and protein docking elements (e.g., FKBP/FRB, Pil1/Aby1, etc.). [236] In some embodiments, a heterologous peptide comprises a protein tag. In some embodiments, the protein tag is referred to as purification tag or a fluorescent protein. In some embodiments, the protein tag is detectable for use in detection of the protein and/or purification of the protein. Accordingly, in some embodiments, compositions, systems and methods comprise a protein tag or use thereof. Any suitable protein tag may be used depending on the purpose of its use. Non-limiting examples of protein tags include a fluorescent protein, a histidine tag, e.g., a 6XHis tag (SEQ ID NO: 109); a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and maltose binding protein (MBP). In some embodiments, the protein tag is a portion of MBP that can be detected and/or purified. Non-limiting examples of fluorescent proteins include green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, and tdTomato. [237] In some embodiments, a heterologous peptide is located at or near the amino terminus (N-terminus) of the protein (e.g., effector protein, effector partner) disclosed herein. In some embodiments, a heterologous peptide is located at or near the carboxy terminus (C-terminus) of the proteins disclosed herein. In some embodiments, a heterologous peptide is located internally in the protein described herein (i.e., is not at the N- or C- terminus of the protein described herein) at a suitable insertion site. [238] In some embodiments, protein (e.g., effector protein or effector partner) described herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more heterologous peptides at or near the N-terminus, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more heterologous peptides at or near the C-terminus, or a combination of these (e.g., one or more heterologous peptides at the amino-terminus and one or more heterologous peptides at the carboxy terminus). When more than one heterologous peptide is present, each may be selected independently of the others, such that a single heterologous peptide may be present in more than one copy and/or in combination with one or more other heterologous peptides present in one or more copies. In some embodiments, a heterologous peptide is considered near the N- or C-terminus when the nearest amino acid of the 53 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT heterologous peptide is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus. [239] In some embodiments, a heterologous peptide described herein comprises a heterologous peptide sequence recited in TABLE 2. In some embodiments, effector proteins described herein comprise an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of the sequences recited in TABLE 1 or a variant thereof and further comprises one or more of the sequences recited in TABLE 2. In some embodiments, a heterologous peptide described herein comprises an effector partner as described en supra. [240] In some embodiments, proteins (e.g., effector protein, effector partner) described herein are encoded by a codon optimized nucleic acid. In some embodiments, a nucleic acid sequence encoding the protein described herein, is codon optimized. In some embodiments, the proteins described herein is codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell. In some embodiments, the effector protein is codon optimized for a human cell. [241] In some embodiments, proteins (e.g., effector protein, effector partner) comprise one or more modifications that provide altered activity as compared to an activity of naturally-occurring counterpart (e.g., a naturally-occurring nuclease or nickase, etc. which is a naturally-occurring protein). In some embodiments, activity (e.g., nickase, nuclease, binding, etc. activity) of proteins described herein is measured relative to a naturally-occurring protein or compositions containing the same in a cleavage assay. [242] For example, proteins (e.g., effector protein, effector partner) comprise one or more modifications that provide increased activity (e.g., catalytic or binding activity) as compared to a naturally-occurring counterpart. As another example, proteins provide increased catalytic activity (e.g., nickase, nuclease, etc. activity) as compared to a naturally-occurring counterpart. In some embodiments, proteins provide enhanced nucleic acid binding activity (e.g., enhanced binding of a guide nucleic acid, and/or target nucleic acid) as compared to a naturally-occurring counterpart. In some embodiments, proteins have a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, or more increase of the activity of a naturally-occurring counterpart. [243] Alternatively or additionally, proteins (e.g., effector protein, effector partner) comprise one or more modifications that reduce the activity (e.g., catalytic or binding activity) of the proteins relative to a naturally-occurring counterpart. In some embodiments, proteins have a 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less decrease of the activity of a naturally-occurring counterpart. In some embodiments, decreased activity comprises decreased catalytic activity (e.g., nickase, nuclease, etc. activity) as compared to a naturally-occurring counterpart. dCAS Proteins 54 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [244] In some embodiments, an effector protein that has decreased catalytic activity is referred to as catalytically or enzymatically inactive, catalytically or enzymatically dead, as a dead protein or a dCas protein. In some embodiments, such a protein comprise an enzymatically inactive domain (e.g. inactive nuclease domain). For example, a nuclease domain (e.g., RuvC domain) of an effector protein, in some embodiments, is deleted or mutated relative to a wildtype counterpart so that it is no longer functional or comprises reduced nuclease activity. In some embodiments, a catalytically inactive effector protein binds to a guide nucleic acid and/or a target nucleic acid but does not cleave the target nucleic acid. In some embodiments, a catalytically inactive effector protein associates with a guide nucleic acid to activate or repress transcription of a target nucleic acid. In some embodiments, a catalytically inactive effector protein is fused to an effector partner that confers an alternative activity to an effector protein activity. Such fusion proteins are described herein and throughout. [245] In some embodiments, catalytically inactive variant effector proteins comprise one or more amino alterations comprising D237A, D418A, D418N, E335A, E335Q, or a combination thereof relative to SEQ ID NO: 1. In some embodiments, catalytically inactive variant effector proteins comprise one or more amino alterations comprising D369A, D369N, D658A, D658N, E567A, E567Q, or a combination thereof relative to SEQ ID NO: 2. Fusion Proteins [246] In some embodiments, compositions, systems, and methods comprise a fusion protein or uses thereof. A fusion protein generally comprises at least one effector protein, at least one effector partner, or a combination thereof. In some embodiments, the effector partner is fused or linked to the effector protein. In some embodiments, the effector partner is fused to the N-terminus of the effector protein. In some embodiments, the effector partner is fused to the C-terminus of the effector protein. [247] In some embodiments, the fusion proteins are multimeric proteins. In some embodiments, the multimeric protein is a homomeric protein. In some embodiments, the multimeric protein is a heteromeric protein. In some embodiments, the fusion protein comprising the effector partner is an effector protein. Accordingly, in such embodiments, the fusion protein can comprise at least two effector proteins that are same. In some embodiments, the fusion protein comprises at least two effector proteins that are different. Unless otherwise indicated, reference to effector proteins throughout the present disclosure include fusion proteins described herein. [248] In some embodiments, the effector partner is a heterologous protein capable of imparting some function or activity that is not provided by an effector protein. In some embodiments, the effector partner is capable of cleaving or modifying the target nucleic acid. In some embodiments, the fusion protein disclosed herein provides cleavage activity, such as cis cleavage activity, trans cleavage activity, nickase activity, nuclease activity, other activity, or a combination thereof. In some embodiments, fusion proteins disclosed herein comprise a RuvC domain capable of cleavage activity. In some embodiments, fusion proteins disclosed herein cleaves nucleic acids, including single stranded RNA (ssRNA), double stranded 55 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT DNA (dsDNA), and single-stranded DNA (ssDNA). In some embodiments, fusion proteins cleave the target nucleic acid at the target sequence or adjacent to the target sequence. [249] In some embodiments, the fusion protein complexes with a guide nucleic acid and the complex interacts with the target nucleic acid. In some embodiments, the interaction comprises one or more of: recognition of a protospacer adjacent motif (PAM) sequence within the target nucleic acid by the effector protein, hybridization of the guide nucleic acid to the target nucleic acid, modification of the target nucleic acid by the fusion protein, or a combination thereof. In some embodiments, recognition of a PAM sequence within a target nucleic acid directs the modification activity of a fusion protein. [250] In some embodiments, modification activity of a fusion protein described herein comprises cleavage activity, binding activity, insertion activity, and substitution activity. In some embodiments, modification activity of an effector protein results in: cleavage of at least one strand of a target nucleic acid, deletion of one or more nucleotides of a target nucleic acid, insertion of one or more nucleotides into a target nucleic acid, substitution of one or more nucleotides of a target nucleic acid with an alternative nucleotide, more than one of the foregoing, or a combination thereof. In some embodiments, an ability of a fusion protein to edit a target nucleic acid depends upon the effector protein being complexed with a guide nucleic acid, the guide nucleic acid being hybridized to a target sequence of the target nucleic acid, the distance between the target sequence and a PAM sequence, or a combination thereof. [251] In some embodiments, the fusion protein described herein comprises a heterologous amino acid sequence that affects formation of a multimeric complex of the fusion protein. By way of non-limiting example, the fusion protein comprises an effector protein described herein and an effector partner comprising a Calcineurin A tag, wherein the fusion protein dimerizes in the presence of Tacrolimus (FK506). Also, by way of non-limiting example, the fusion protein comprises an effector protein described herein and a SpyTag configured to dimerize or associate with another effector protein in a multimeric complex. Multimeric complex formation is further described herein. Multimeric Complexes [252] Compositions, systems, and methods of the present disclosure comprise a multimeric complex or uses thereof, wherein the multimeric complex comprises one or more effector proteins that non-covalently interact with one another. In some embodiments, a multimeric complex comprises enhanced activity relative to the activity of any one of its effector proteins alone. For example, in some embodiments, a multimeric complex comprises two effector proteins (e.g., in dimeric form), wherein the multimeric complex comprises greater nucleic acid binding affinity and/or nuclease activity than that of either of the effector proteins provided in monomeric form. In some embodiments, a multimeric complex comprises one or more heterologous proteins fused to one or more effector proteins, wherein the fusion proteins are capable of different activity than that of the one or more effector proteins. In another example, a multimeric complex comprises an effector protein and a partner protein, wherein the multimeric complex comprises an effector partner, and wherein the multimeric complex comprises greater nucleic acid binding affinity 56 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT and/or nuclease activity than that of either of the effector protein or effector partner provided in monomeric form. In some embodiments, a multimeric complex comprises an affinity for a target sequence of a target nucleic acid and is capable of catalytic activity (e.g., cleaving, nicking, inserting or otherwise editing the nucleic acid) at or near the target sequence. In some embodiments, a multimeric complex comprises an affinity for a donor nucleic acid and is capable of catalytic activity (e.g., cleaving, nicking, editing or otherwise modifying the nucleic acid by creating cuts) at or near one or more ends of the donor nucleic acid. In some embodiments, multimeric complexes are active when complexed with a guide nucleic acid. In some embodiments, multimeric complexes are active when complexed with a target nucleic acid. In some embodiments, multimeric complexes are active when complexed with a guide nucleic acid, a target nucleic acid, and/or a donor nucleic acid. In some embodiments, the multimeric complex cleaves the target nucleic acid. In some embodiments, the multimeric complex nicks the target nucleic acid. [253] Various aspects of the present disclosure include compositions and methods comprising multiple polypeptides (e.g., effector proteins, effector partners, fusion proteins, or a combination thereof), and uses thereof, respectively. An effector protein comprising at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to any one of the effector protein sequences of TABLE 1 or a variant thereof are provided with a second effector protein. In some embodiments, two effector proteins target different nucleic acid sequences. Two effector proteins may target different types of nucleic acids (e.g., a first effector protein targets double- and single-stranded nucleic acids, and a second effector protein only targets single-stranded nucleic acids). It is understood that when discussing the use of more than one effector protein in compositions, systems, and methods provided herein, the multimeric complex form is also described. [254] In some embodiments, multimeric complexes comprise at least one polypeptide (e.g., effector protein, effector partner, or fusion protein) as described herein. In some embodiments, the multimeric complex is a dimer comprising a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide and the second polypeptide comprise identical amino acid sequences. In some embodiments, the first polypeptide and the second polypeptide comprise amino acid sequences that are at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to each other. In some embodiments, the first polypeptide and the second polypeptide comprise similar amino acid sequences. In some embodiments, the first polypeptide and the second polypeptide comprise amino acid sequences that are at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% similar to each other. [255] In some embodiments, the multimeric complex is a heterodimeric complex comprising at least two polypeptides (e.g., effector proteins, effector partners, fusion proteins, or a combination thereof) of different amino acid sequences. In some embodiments, the multimeric complex comprises two, three, four, five, six, seven, eight, nine, or ten polypeptides. In some embodiments, the multimeric complex is a heterodimeric complex comprising a first effector protein and a second effector protein, wherein the amino acid sequence of the first effector protein is less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, 57 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% identical to the amino acid sequence of the second effector protein. [256] In some embodiments, at least one effector protein of the multimeric complex comprises an amino acid sequence with at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identity to any one of the effector protein sequences of TABLE 1 or a variant thereof . In some embodiments, each effector protein of the multimeric complex independently comprises an amino acid sequence with at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identity to any one of the effector protein sequences of TABLE 1 or a variant thereof . [257] In some embodiments, the multimeric complex described herein is capable of targeting polyA signals, splice site acceptors, and start codons. In some embodiments, the multimeric complex cannot create stop codons for knock-down. In some embodiments, the multimeric complex is a dimer comprising fusion protein described herein. In some embodiments, the fusion protein comprises the effector protein described herein and the effector partner described herein. In some embodiments, the dimer is formed due to non- covalent interactions between the effector proteins of monomers. In some embodiments, N- and C- termini of “formerly active” monomer is closer to 5’ region of non-target strand, while the termini of the “other” monomer is closer to 3’ region, which results in a larger editing window of the multimeric complex having a larger editing window on the non-target strand. In some embodiments, the multimeric complex has a lower editing window for a target strand due to in accessibility for the effector partner. Synthesis, Isolation and Assaying [258] Polypeptides (e.g., effector proteins, effector partners, and fusion proteins) of the present disclosure are synthesized, using any suitable method. In some embodiments, the polypeptides are produced in vitro or by eukaryotic cells or by prokaryotic cells. In some embodiments, the polypeptides are further processed by unfolding (e.g. heat denaturation, dithiothreitol reduction, etc.) and are further refolded, using any suitable method. In some embodiments, the nucleic acid(s) encoding the polypeptides described herein, the recombinant nucleic acid(s) described herein, the vectors described herein are produced in vitro or in vivo by eukaryotic cells or by prokaryotic cells. [259] Any suitable method of generating and assaying the polypeptides (e.g., effector proteins, effector partners, and fusion proteins) described herein are used. Such methods include, but are not limited to, site- directed mutagenesis, random mutagenesis, combinatorial libraries, and other mutagenesis methods described herein (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Gillman et al., Directed Evolution Library Creation: Methods and Protocols (Methods in Molecular Biology) Springer, 2nd ed (2014)). One non-limiting example of a method for preparing the polypeptide is to express recombinant nucleic acids encoding the polypeptide in a suitable 58 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT microbial organism, such as a bacterial cell, a yeast cell, or other suitable cell, using methods well known in the art. Exemplary methods are also described in the Examples provided herein. [260] In some embodiments, a polypeptide provided herein is an isolated polypeptide (e.g., effector protein, effector partner, and fusion protein). In some embodiments, the polypeptide is isolated and purified for use in compositions, systems, and/or methods described herein. In some embodiments, methods described here comprise the step of isolating polypeptides described herein. Any suitable method to provide isolated polypeptides described herein is used in the present disclosure, for example, recombinant expression systems, precipitation, gel filtration, ion-exchange, reverse-phase and affinity chromatography. Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzymology, Vol. 182, (Academic Press, (1990)). Alternatively, the isolated polypeptides of the present disclosure can be obtained using well-known recombinant methods (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). The methods and conditions for biochemical purification of a polypeptide described herein can be chosen by those skilled in the art, and purification monitored, for example, by a functional assay. [261] In some embodiments, compositions, systems, and methods described herein further comprise a purification tag that can be attached to a polypeptide (e.g., effector protein, effector partner, and fusion protein), or a nucleic acid encoding the purification tag that can be attached to a nucleic acid encoding the polypeptide as described herein. In some embodiments, the purification tag comprises an amino acid sequence which can attach or bind with high affinity to a separation substrate and assist in isolating the polypeptide of interest from its environment, which comprises its biological source, such as a cell lysate. Attachment of the purification tag is at the N or C terminus of the polypeptide. Furthermore, an amino acid sequence recognized by a protease or a nucleic acid encoding for an amino acid sequence recognized by a protease, such as TEV protease or the HRV3C protease, is inserted between the purification tag and the polypeptide, such that biochemical cleavage of the sequence with the protease after initial purification liberates the purification tag. In some embodiments, purification and/or isolation are performed through high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. Non-limiting examples of purification tags are as described herein. [262] In some embodiments, polypeptides (e.g., effector proteins, effector partners, and fusion proteins) described herein are isolated from cell lysate. In some embodiments, the compositions described herein comprise 20% or more by weight, 75% or more by weight, 95% or more by weight, or 99.5% or more by weight of the polypeptide, related to the method of preparation of compositions described herein and its purification thereof, wherein percentages refer to total polypeptide content relative to contaminants. Thus, in some embodiments, the polypeptide is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants, non-engineered proteins or other macromolecules, etc.). 59 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT Protospacer Adjacent Motif (PAM) Sequences [263] In some embodiments, polypeptide (e.g., effector protein, effector partner, and fusion protein) of the present disclosure cleaves or nicks a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some embodiments, the target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand. In some embodiments, cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides of a 5’ or 3’ terminus of a PAM sequence. In some embodiments, polypeptides described herein recognize a PAM sequence. In some embodiments, recognizing a PAM sequence comprises interacting with a sequence adjacent to the PAM. In some embodiments, a target nucleic acid comprises a target sequence that is adjacent to a PAM sequence. In some embodiments, the polypeptide does not require a PAM to bind and/or cleave a target nucleic acid. [264] In some embodiments, a target nucleic acid is a single stranded target nucleic acid comprising a target sequence. Accordingly, in some embodiments, the single stranded target nucleic acid comprises a PAM sequence described herein that is adjacent (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides) or directly adjacent to the target sequence. In some embodiments, an RNP cleaves the single stranded target nucleic acid. [265] In some embodiments, a target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand, wherein the target strand comprises a target sequence. In some embodiments, the PAM sequence is located on the target strand. In some embodiments, the PAM sequence is located on the non-target strand. In some embodiments, the PAM sequence described herein is adjacent (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides) to the target sequence on the target strand or the non-target strand. In some embodiments, the PAM sequence is located 5’ of a reverse complement of the target sequence on the non-target strand. In some embodiments, such a PAM described herein is directly adjacent to the target sequence on the target strand or the non-target strand. In some embodiments, an RNP cleaves the target strand or the non-target strand. In some embodiments, the RNP cleaves both, the target strand and the non-target strand. In some embodiments, an RNP recognizes the PAM sequence, and hybridizes to a target sequence of the target nucleic acid. In some embodiments, the RNP cleaves the target nucleic acid, wherein the RNP has recognized the PAM sequence and is hybridized to the target sequence. [266] In some embodiments, an effector protein described herein, or a multimeric complex thereof, recognizes a PAM on a target nucleic acid. In some embodiments, multiple effector proteins of the multimeric complex recognize a PAM on a target nucleic acid. In some embodiments, at least two of the multiple effector proteins recognize the same PAM sequence. In some embodiments, at least two of the multiple effector proteins recognize different PAM sequences. In some embodiments, only one effector protein of the multimeric complex recognizes a PAM on a target nucleic acid. 60 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [267] In some embodiments, an effector protein of the present disclosure, or a multimeric complex thereof, cleaves or nicks a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some embodiments, cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of a 5’ or 3’ terminus of a PAM sequence. [268] In some embodiments, a PAM sequence provided herein comprises any one of the nucleotide sequences recited in TABLE 3. In some embodiments, an effector protein described herein recognizes a PAM sequence comprising any one of the sequences recited in TABLE 3, and wherein optionally the PAM sequence is adjacent to a target sequence of a target nucleic acid. Nucleic Acid Systems Guide Nucleic Acids [269] The compositions, systems, and methods of the present disclosure comprise a guide nucleic acid or a use thereof. Unless otherwise indicated, compositions, systems and methods comprising guide nucleic acids or uses thereof, as described herein and throughout, include DNA molecules, such as expression vectors, that encode a guide nucleic acid. Accordingly, compositions, systems, and methods of the present disclosure comprise a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid. Guide nucleic acids are also referred to herein as “guide RNA.” A guide nucleic acid, as well as any components thereof (e.g., spacer sequence, repeat sequence, linker nucleotide sequence, handle sequence, intermediary sequence etc.) comprise one or more deoxyribonucleotides, ribonucleotides, biochemically or chemically modified nucleotides (e.g., one or more engineered modifications as described herein), or a combination thereof. [270] In some embodiments, a guide nucleic acid comprises a naturally occurring sequence. In some embodiments, a guide nucleic acid comprises a non-naturally occurring sequence, wherein the sequence of the guide nucleic acid, or any portion thereof, is different from the sequence of a naturally occurring guide nucleic acid. A guide nucleic acid of the present disclosure comprises one or more of the following: a) a single nucleic acid molecule; b) a DNA base; c) an RNA base; d) a modified base; e) a modified sugar; and f) a modified backbone. In some embodiments, the guide nucleic acid comprises one or more phosphorothioate (PS) backbone modifications, 2’-fluoro (2’-F) sugar modifications, or 2’-O-Methyl (2’OMe) sugar modification. Modifications are described herein and throughout the present disclosure (e.g., in the section entitled “Engineered Modifications”). In some embodiments, a guide nucleic acid is chemically synthesized or recombinantly produced by any suitable methods. In some embodiments, guide nucleic acids and portions thereof are found in or identified from a CRISPR array present in the genome of a host organism or cell. [271] In general, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the target sequence. In some embodiments, the guide nucleic acid comprises at least 10 contiguous nucleotides that are complementary to the target sequence in the target nucleic acid. In some embodiments, guide nucleic acid 61 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the target sequence. [272] In general, a guide nucleic acid comprises a first region that is not complementary to a target sequence of a target nucleic acid (FR) and a second region is complementary to the target sequence of the target nucleic acid (SR), wherein the FR and the SR are heterologous to each other. In some embodiments, FR is located 5’ to SR (FR-SR). In some embodiments, SR is located 5’ to FR (SR-FR). In some embodiments, the FR comprises one or more repeat sequence, handle sequence, intermediary sequence, or a combination thereof. In some embodiments, at least a portion of the FR interacts or binds to an effector protein. In some embodiments, the SR comprises a spacer sequence, wherein the spacer sequence can interact in a sequence-specific manner with (e.g., has complementarity with, or can hybridize to a target sequence in) a target nucleic acid. [273] In some embodiments, the first region, the second region, or both are about 8 nucleotides, about 10 nucleotides, about 12 nucleotides, about 14 nucleotides, about 16 nucleotides, about 18 nucleotides, about 20 nucleotides, about 22 nucleotides, about 24 nucleotides, about 26 nucleotides, about 28 nucleotides, about 30 nucleotides, about 32 nucleotides, about 34 nucleotides, about 36 nucleotides, about 38 nucleotides, about 40 nucleotides, about 42 nucleotides, about 44 nucleotides, about 46 nucleotides, about 48 nucleotides, or about 50 nucleotides long. [274] In some embodiments, the first region, the second region, or both are from about 8 to about 12, from about 8 to about 16, from about 8 to about 20, from about 8 to about 24, from about 8 to about 28, from about 8 to about 30, from about 8 to about 32, from about 8 to about 34, from about 8 to about 36, from about 8 to about 38, from about 8 to about 40, from about 8 to about 42, from about 8 to about 44, from about 8 to about 48, or from about 8 to about 50 nucleotides long. [275] In some embodiments, the first region, the second region, or both comprise a GC content of about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%. In some embodiments, the first region, the second region, or both comprise a GC content of from about 1% to about 95%, from about 5% to about 90%, from about 10% to about 80%, from about 15% to about 70%, from about 20% to about 60%, from about 25% to about 50%, or from about 30% to about 40%. [276] In some embodiments, the first region, the second region, or both have a melting temperature of about 38 °C, about 40 °C, about 42 °C, about 44 °C, about 46 °C, about 48 °C, about 50 °C, about 52 °C, about 54 °C, about 56 °C, about 58 °C, about 60 °C, about 62 °C, about 64 °C, about 66 °C, about 68 °C, about 70 °C, about 72 °C, about 74 °C, about 76 °C, about 78 °C, about 80 °C, about 82 °C, about 84 °C, about 86 °C, about 88 °C, about 90 °C, or about 92 °C. In some embodiments, the first region, the second region, or both have a melting temperature of from about 35 °C to about 40 °C, from about 35 °C to about 45 °C, from about 35 °C to about 50 °C, from about 35 °C to about 55 °C, from about 35 °C to about 60 62 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT °C, from about 35 °C to about 65 °C, from about 35 °C to about 70 °C, from about 35 °C to about 75 °C, from about 35 °C to about 80 °C, or from about 35 °C to about 85 °C. [277] In some embodiments, the compositions, systems, and methods of the present disclosure further comprise an additional nucleic acid, wherein a portion of the additional nucleic acid at least partially hybridizes to the first region of the guide nucleic acid. In some embodiments, the additional nucleic acid is at least partially hybridized to the 5’ end of the second region of the guide nucleic acid. In some embodiments, an unhybridized portion of the additional nucleic acid, at least partially, interacts with an effector protein or polypeptide. In some embodiments, the compositions, systems, and methods of the present disclosure comprise a dual nucleic acid system comprising the guide nucleic acid and the additional nucleic acid as described herein. [278] In some embodiments, the guide nucleic acid also forms complexes as described through herein. For example, in some embodiments, a guide nucleic acid hybridizes to another nucleic acid, such as target nucleic acid, or a portion thereof. In another example, a guide nucleic acid complexes with an effector protein. In such embodiments, a guide nucleic acid-effector protein complex is described herein as an RNP. In some embodiments, when in a complex, at least a portion of the complex binds, recognizes, and/or hybridizes to a target nucleic acid. For example, when a guide nucleic acid and an effector protein are complexed to form an RNP, at least a portion of the guide nucleic acid hybridizes to a target sequence in a target nucleic acid. Those skilled in the art in reading the below specific examples of guide nucleic acids as used in RNPs described herein, will understand that in some embodiments, a RNP hybridizes to one or more target sequences in a target nucleic acid, thereby allowing the RNP to modify and/or recognize a target nucleic acid or sequence contained therein (e.g., PAM) or to modify and/or recognize non-target sequences depending on the guide nucleic acid, and in some embodiments, the effector protein, used. [279] In some embodiments, a guide nucleic acid comprises or forms intramolecular secondary structure (e.g., hairpins, stem-loops, etc.). In some embodiments, a guide nucleic acid comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the guide nucleic acid comprises a pseudoknot (e.g., a secondary structure comprising a stem, at least partially, hybridized to a second stem or half-stem secondary structure). In some embodiments, an effector protein recognizes a guide nucleic acid comprising multiple stem regions. In some embodiments, the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the guide nucleic acid comprises at least 2, at least 3, at least 4, or at least 5 stem regions. [280] In some embodiments, the compositions, systems, and methods of the present disclosure comprise two or more guide nucleic acids (e.g., 2, 3, 4, 5, 6, 7, 9, 10 or more guide nucleic acids), and/or uses thereof. In some embodiments, multiple guide nucleic acids target an effector protein to different locations in the target nucleic acid by hybridizing to different target sequences. In some embodiments, a first guide nucleic 63 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT acid hybridizes within a location of the target nucleic acid that is different from where a second guide nucleic acid hybridizes the target nucleic acid. In some embodiments, the first loci and the second loci of the target nucleic acid are located at least 1, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 nucleotides apart. In some embodiments, the first loci and the second loci of the target nucleic acid are located between 100 and 200, 200 and 300, 300 and 400, 400 and 500, 500 and 600, 600 and 700, 700 and 800, 800 and 900 or 900 and 1000 nucleotides apart. In some embodiments, the first loci and/or the second loci of the target nucleic acid are located in an intron of a gene. In some embodiments, the first loci and/or the second loci of the target nucleic acid are located in an exon of a gene. In some embodiments, the first loci and/or the second loci of the target nucleic acid span an exon-intron junction of a gene. In some embodiments, the first portion and/or the second portion of the target nucleic acid are located on either side of an exon and cutting at both sites results in deletion of the exon. In some embodiments, composition, systems, and methods comprise a donor nucleic acid that is inserted in replacement of a deleted or cleaved sequence of the target nucleic acid. In some embodiments, compositions, systems, and methods comprising multiple guide nucleic acids or uses thereof comprise multiple effector proteins, wherein the effector proteins is identical, non-identical, or a combination thereof. [281] In some embodiments, a guide nucleic acid comprises about: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 linked nucleotides. In general, a guide nucleic acid comprises at least: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 linked nucleotides. In some embodiments, the guide nucleic acid has about 10 to about 60, about 20 to about 50, or about 30 to about 40 linked nucleotides. [282] In some embodiments, a guide nucleic acid comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary to a eukaryotic sequence. Such a eukaryotic sequence is a nucleotide sequence that is present in a host eukaryotic cell. Such a nucleotide sequence is distinguished from nucleotide sequences present in other host cells, such as prokaryotic cells, or viruses. Said sequences present in a eukaryotic cell can be located in a gene, an exon, an intron, a non-coding (e.g., promoter or enhancer) region, a selectable marker, tag, or signal. In some embodiments, a target sequence is a eukaryotic sequence. [283] In some embodiments, a length of a guide nucleic acid is about 30 to about 120 linked nucleotides. In some embodiments, the length of a guide nucleic acid is about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 90, about 50 to about 80, about 50 to about 70, or about 50 to about 60 linked nucleotides. In some embodiments, the length of a guide nucleic acid is about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides. In some embodiments, the length of a guide nucleic acid is greater than about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides. In some embodiments, the length of a guide nucleic acid is not greater than about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 64 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, or about 125 linked nucleotides. [284] In some embodiments, guide nucleic acids comprise additional elements that contribute additional functionality (e.g., stability, heat resistance, etc.) to the guide nucleic acid. In some embodiments, the elements comprise one or more nucleotide alterations, nucleotide sequences, intermolecular secondary structures, or intramolecular secondary structures (e.g., one or more hair pin regions, one or more bulges, etc.). [285] In some embodiments, guide nucleic acids comprise one or more linkers connecting different nucleotide sequences as described herein. In some embodiments, a linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides. In some embodiments, a linker comprises any suitable linker, examples of which are described herein. [286] In some embodiments, guide nucleic acids comprise one or more nucleotide sequences as described herein (e.g., TABLE 4, TABLE 5, TABLE 6, TABLE 7, and TABLE 8). In some embodiments, the nucleotide sequences described herein (e.g., TABLE 4, TABLE 5, TABLE 6, TABLE 7, and TABLE 8) are described as a nucleotide sequence of either DNA or RNA, however, no matter the form of the sequence described, it is readily understood that such nucleotide sequences may be revised to be RNA or DNA, as needed, for describing a sequence within a guide nucleic acid itself or the sequence that encodes a guide nucleic acid, such as a nucleotide sequence described herein for a vector. Similarly, disclosure of the nucleotide sequences described herein (e.g., TABLE 4, TABLE 5, TABLE 6, TABLE 7, and TABLE 8) also discloses the complementary nucleotide sequence, the reverse nucleotide sequence, and the reverse complement nucleotide sequence, any one of which may be a nucleotide sequence for use in a guide nucleic acid as described herein. In some embodiments, guide nucleic acid sequence(s) comprises one or more nucleotide alterations at one or more positions in any one of the sequences described herein. Alternative nucleotides may be any one or more of A, C, G, T or U, or a deletion, or an insertion. [287] In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is capable of hybridizing to a target sequence in a target nucleic acid, wherein the target nucleic acid is any one of: a naturally occurring eukaryotic sequence, a naturally occurring prokaryotic sequence, a naturally occurring viral sequence, a naturally occurring bacterial sequence, a naturally occurring fungal sequence, an engineered eukaryotic sequence, an engineered prokaryotic sequence, an engineered viral sequence, an engineered bacterial sequence, an engineered fungal sequence, a fragment of a naturally occurring sequence, a fragment of an engineered sequence, and a combination thereof. [288] In some embodiments, the guide nucleic acid is isolated from any one of: a naturally occurring cell, a eukaryotic cell, a prokaryotic cell, a plant cell, a fungal cell, an animal cell, cell of an invertebrate, a fly cell, a cell of a vertebrate, a mammalian cell, a primate cell, a non-human primate cell, a human cell, a living cell, a non-living cell, a modified cell, a derived cell, and a non-naturally occurring cell. Repeat Sequence 65 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [289] In some embodiments, guide nucleic acids described herein comprise one or more repeat sequences. In some embodiments, a repeat sequence comprises a nucleotide sequence that is not complementary to a target sequence of a target nucleic acid. In some embodiments, a repeat sequence comprises a nucleotide sequence that interacts with an effector protein. In some embodiments, a repeat sequence is connected to another sequence of a guide nucleic acid, such as an intermediary sequence, that is capable of non-covalently interacting with an effector protein. In some embodiments, a repeat sequence includes a nucleotide sequence that is capable of forming a guide nucleic acid-effector protein complex (e.g., a RNP complex). [290] In some embodiments, the repeat sequence is between 10 and 50, 12 and 48, 14 and 46, 16 and 44, and 18 and 42 nucleotides in length. [291] In some embodiments, a repeat sequence is adjacent to a spacer sequence. In some embodiments, a repeat sequence is followed by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is preceded by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is adjacent to an intermediary sequence. In some embodiments, a repeat sequence is 3’ to an intermediary sequence. In some embodiments, an intermediary sequence is followed by a repeat sequence, which is followed by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is linked to a spacer sequence and/or an intermediary sequence. In some embodiments, a guide nucleic acid comprises a repeat sequence linked to a spacer sequence and/or to an intermediary sequence by a direct link or by any suitable linker, examples of which are described herein. [292] In some embodiments, guide nucleic acids comprise more than one repeat sequence (e.g., two or more, three or more, or four or more repeat sequences). In some embodiments, a guide nucleic acid comprises more than one repeat sequence separated by another sequence of the guide nucleic acid. For example, in some embodiments, a guide nucleic acid comprises two repeat sequences, wherein the first repeat sequence is followed by a spacer sequence, and the spacer sequence is followed by a second repeat sequence in the 5’ to 3’ direction. In some embodiments, the more than one repeat sequences are identical. In some embodiments, the more than one repeat sequences are not identical. [293] In some embodiments, the repeat sequence comprises two sequences that are complementary to each other and hybridize to form a double stranded RNA duplex (dsRNA duplex). In some embodiments, the two sequences are not directly linked and hybridize to form a stem loop structure. In some embodiments, the dsRNA duplex comprises 5, 10, 15, 20 or 25 base pairs (bp). In some embodiments, not all nucleotides of the dsRNA duplex are paired, and therefore the duplex forming sequence comprises a bulge. In some embodiments, the repeat sequence comprises a hairpin or stem-loop structure, optionally at the 5’ portion of the repeat sequence. In some embodiments, a strand of the stem portion comprises a sequence and the other strand of the stem portion comprises a sequence that is, at least partially, complementary. In some embodiments, such sequences comprise 65% to 100% complementarity (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementarity). In some embodiments, a guide nucleic acid comprises a nucleotide sequence that, when involved in hybridization events, hybridizes over one or more segments of a target 66 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.). [294] In some embodiments, a repeat sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to an equal length portion of any one of the repeat sequences in TABLE 4. In some embodiments, the repeat sequence is at least 85% identical to any one of sequences recited in TABLE 4. In some embodiments, a repeat sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides of any one of the nucleotide sequences recited in TABLE 4. [295] In some embodiments, a repeat sequence comprises one or more nucleotide alterations at one or more positions in the nucleotide sequence recited in TABLE 4. Alternative nucleotides can be any one or more of A, C, G, T or U, or a deletion, or an insertion. Spacer Sequence [296] In some embodiments, guide nucleic acids described herein comprise one or more spacer sequences. In some embodiments, a spacer sequence is capable of hybridizing to a target sequence of a target nucleic acid. In some embodiments, a spacer sequence comprises a nucleotide sequence that is, at least partially, hybridizable to an equal length of a sequence (e.g., a target sequence) of a target nucleic acid. Exemplary hybridization conditions are described herein. In some embodiments, the spacer sequence functions to direct an RNP complex comprising the guide nucleic acid to the target nucleic acid for detection and/or modification. In some embodiments, the spacer sequence functions to direct a RNP to the target nucleic acid for detection and/or modification. In some embodiments, a spacer sequence is complementary to a target sequence that is adjacent to a PAM that is recognizable by an effector protein described herein. [297] In some embodiments, a spacer sequence comprises at least 5 to about 50 contiguous nucleotides that are complementary to a target sequence in a target nucleic acid. In some embodiments, a spacer sequence comprises at least 5 to about 50 linked nucleotides. In some embodiments, a spacer sequence comprises at least 5 to about 50, at least 5 to about 25, at least about 10 to about 25, or at least 15 to about 25 linked nucleotides. In some embodiments, the spacer sequence comprises 15-28 linked nucleotides. In some embodiments, a spacer sequence comprises 15-26, 15-24, 15-22, 15-20, 15-18, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 17-26, 17-24, 17-22, 17-20, 17-18, 18-26, 18-24, or 18-22 linked nucleotides. In some embodiments, the spacer sequence comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides. [298] In some embodiments, a spacer sequence is adjacent to a repeat sequence. In some embodiments, a spacer sequence follows a repeat sequence in a 5’ to 3’ direction. In some embodiments, a spacer sequence precedes a repeat sequence in a 5’ to 3’ direction. In some embodiments, the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present within the same molecule. In some embodiments, 67 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT the spacer(s) and repeat sequence(s) are linked directly to one another. In some embodiments, a linker is present between the spacer(s) and repeat sequences. In some embodiments, linkers comprise any suitable linker. In some embodiments, the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present in separate molecules, which are joined to one another by base pairing interactions. [299] In some embodiments, a spacer sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of a target nucleic acid. A spacer sequence is capable of hybridizing to an equal length portion of a target nucleic acid (e.g., a target sequence). In some embodiments, a target nucleic acid, such as DNA or RNA, comprises a cancer gene or gene associated with a genetic disorder, or an amplicon thereof, as described herein. In some embodiments, a target nucleic acid is a gene selected from TABLE 9. In some embodiments, a spacer sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of a target nucleic acid selected from TABLE 9. In some embodiments, a target nucleic acid is a nucleic acid associated with a disease or syndrome recited in TABLE 10. In some embodiments, a spacer sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of a target nucleic acid associated with a disease or syndrome recited in TABLE 10. In some embodiments, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are capable of hybridizing to the target sequence. In some embodiments, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary to the target sequence. [300] It is understood that the spacer sequence of a spacer sequence need not be 100% complementary to that of a target sequence of a target nucleic acid to hybridize or hybridize specifically to the target sequence. For example, the spacer sequence, in some embodiments, comprises at least one alteration, such as a substituted or modified nucleotide, that is not complementary to the corresponding nucleotide of the target sequence. Spacer sequences are further described throughout herein, for example, in the Examples section. [301] In some embodiments, a spacer sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the spacer sequences in Example 6 or 7. In some embodiments, the spacer sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 , or at least 21 contiguous nucleotides of any one of the sequences recited in Example 6 or 7. [302] In some embodiments, a spacer sequence is capable of hybridizing to a target sequence of a target nucleic acid (e.g., DMPK gene). In some embodiments, a spacer sequence comprises a nucleotide sequence that is, at least partially, hybridizable to an equal length of a sequence (e.g., a target sequence within the DMPK gene) of a target nucleic acid. 68 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [303] In some embodiments, additional guide nucleic acids described herein comprise one or more spacer sequences targeting different sequences in a target nucleic acid (e.g., DMPK gene), as compared to the one or more spacer sequences of the guide nucleic acid. Linker for Nucleic Acids [304] In some embodiments, a guide nucleic acid for use with compositions, systems, and methods described herein comprises one or more linkers, or a nucleic acid encoding one or more linkers. In some embodiments, the guide nucleic acid comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten linkers. In some embodiments, the guide nucleic acid comprises one, two, three, four, five, six, seven, eight, nine, or ten linkers. In some embodiments, the guide nucleic acid comprises more than one linker. In some embodiments, at least two of the more than one linker are the same. In some embodiments, at least two of the more than one linker are not same. [305] In some embodiments, a linker comprises one to ten, one to seven, one to five, one to three, two to ten, two to eight, two to six, two to four, three to ten, three to seven, three to five, four to ten, four to eight, four to six, five to ten, five to seven, six to ten, six to eight, seven to ten, or eight to ten linked nucleotides. In some embodiments, the linker comprises one, two, three, four, five, six, seven, eight, nine, or ten linked nucleotides. In some embodiments, a linker comprises a nucleotide sequence of 5’-GAAA-3’. [306] In some embodiments, a guide nucleic acid comprises one or more linkers connecting one or more repeat sequences. In some embodiments, the guide nucleic acid comprises one or more linkers connecting one or more repeat sequences and one or more spacer sequences. In some embodiments, the guide nucleic acid comprises at least two repeat sequences connected by a linker. Intermediary sequence [307] In some embodiments, guide nucleic acids described herein comprise one or more intermediary sequences. In general, an intermediary sequence used in the present disclosure is not transactivated or transactivating. In some embodiments, an intermediary sequence is also be referred to as an intermediary RNA, although it may comprise deoxyribonucleotides instead of or in addition to ribonucleotides, and/or modified bases. In general, the intermediary sequence non-covalently binds to an effector protein. In some embodiments, the intermediary sequence forms a secondary structure, for example in a cell, and an effector protein binds the secondary structure. [308] In some embodiments, a length of the intermediary sequence is at least 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, a length of the intermediary sequence is not greater than 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, the length of the intermediary sequence is about 30 to about 210, about 60 to about 210, about 90 to about 210, about 120 to about 210, about 150 to about 210, about 180 to about 210, about 30 to about 180, about 60 to about 180, about 90 to about 180, about 120 to about 180, or about 150 to about 180 linked nucleotides. 69 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [309] In some embodiments, an intermediary sequence also comprises or forms a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of an effector protein to a guide nucleic acid and/or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region). In some embodiments, an intermediary sequence comprises from 5’ to 3’, a 5’ region, a hairpin region, and a 3’ region. In some embodiments, the 5’ region hybridizes to the 3’ region. In some embodiments, the 5’ region of the intermediary sequence does not hybridize to the 3’ region. [310] In some embodiments, the hairpin region comprises a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first sequence and the second sequence. In some embodiments, an intermediary sequence comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, an intermediary sequence comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). In some embodiments, an effector protein interacts with an intermediary sequence comprising a single stem region or multiple stem regions. In some embodiments, the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, an intermediary sequence comprises 1, 2, 3, 4, 5 or more stem regions. [311] In some embodiments, an intermediary sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the intermediary sequences in TABLE 6 or TABLE 8. In some embodiments, an intermediary sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, or at least 140 contiguous nucleotides of any one of the intermediary sequences recited in TABLE 6 or TABLE 8. Handle Sequence [312] In some embodiments, guide nucleic acids described herein comprise one or more handle sequences. In some embodiments, the handle sequence comprises an intermediary sequence. In such instances, at least a portion of an intermediary sequence non-covalently bonds with an effector protein. In some embodiments, the intermediary sequence is at the 3’-end of the handle sequence. In some embodiments, the intermediary sequence is at the 5’- end of the handle sequence. Additionally, or alternatively, in some embodiments, the handle sequence further comprises one or more of linkers and repeat sequences. In such instances, at least a portion of an intermediary sequence, or both of at least a portion of the intermediary sequence and at least a portion of repeat sequence, non-covalently interacts with an effector protein. In some embodiments, an intermediary sequence and repeat sequence are directly linked (e.g., covalently linked, such as through a phosphodiester bond). In some embodiments, the intermediary 70 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT sequence and repeat sequence are linked by a suitable linker, examples of which are provided herein. In some embodiments, the linker comprises a sequence of 5’-GAAA-3’. In some embodiments, the intermediary sequence is 5’ to the repeat sequence. In some embodiments, the intermediary sequence is 5’ to the linker. In some embodiments, the intermediary sequence is 3’ to the repeat sequence. In some embodiments, the intermediary sequence is 3’ to the linker. In some embodiments, the repeat sequence is 3’ to the linker. In some embodiments, the repeat sequence is 5’ to the linker. In general, a single guide nucleic acid, also referred to as a single guide RNA (sgRNA), comprises a handle sequence comprising an intermediary sequence, and optionally one or more of a repeat sequence and a linker. [313] In some embodiments, a handle sequence comprises or forms a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of an effector protein to a guide nucleic acid and/or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region). In some embodiments, handle sequences comprise a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the handle sequence comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). In some embodiments, an effector protein recognizes a handle sequence comprising multiple stem regions. In some embodiments, the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the handle sequence comprises at least 2, at least 3, at least 4, or at least 5 stem regions. [314] In some embodiments, a length of the handle sequence is at least 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, a length of the handle sequence is not greater than 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, the length of the handle sequence is about 30 to about 210, about 60 to about 210, about 90 to about 210, about 120 to about 210, about 150 to about 210, about 180 to about 210, about 30 to about 180, about 60 to about 180, about 90 to about 180, about 120 to about 180, or about 150 to about 180 linked nucleotides. [315] In some embodiments, a handle sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the handle sequences in TABLE 6. In some embodiments, a handle sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, or at least 140 contiguous nucleotides of any one of the handle sequences recited in TABLE 6. A Single Nucleic Acid System 71 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [316] In some embodiments, compositions, systems and methods described herein comprise a single nucleic acid system comprising a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid, and one or more effector proteins or a nucleotide sequence encoding the one or more effector proteins. In some embodiments, a first region (FR) of the guide nucleic acid non-covalently interacts with the one or more polypeptides described herein. In some embodiments, a second region (SR) of the guide nucleic acid hybridizes with a target sequence of the target nucleic acid. In the single nucleic acid system having a complex of the guide nucleic acid and the effector protein, the effector protein is not transactivated by the guide nucleic acid. In other words, activity of effector protein does not require binding to a second non- target nucleic acid molecule. An exemplary guide nucleic acid for a single nucleic acid system is a crRNA or an sgRNA. crRNA [317] In some embodiments, a guide nucleic acid comprises a crRNA. In some embodiments, the guide nucleic acid is the crRNA. In general, a crRNA comprises a first region (FR) and a second region (SR), wherein the FR of the crRNA comprises a repeat sequence, and the SR of the crRNA comprises a spacer sequence. In some embodiments, the repeat sequence and the spacer sequences are directly connected to each other (e.g., covalent bond (phosphodiester bond)). In some embodiments, the repeat sequence and the spacer sequence are connected by a linker. [318] In some embodiments, a crRNA is useful as a single nucleic acid system for compositions, methods, and systems described herein or as part of a single nucleic acid system for compositions, methods, and systems described herein. In some embodiments, a crRNA is useful as part of a single nucleic acid system for compositions, methods, and systems described herein. In such embodiments, a single nucleic acid system comprises a guide nucleic acid comprising a crRNA wherein, a repeat sequence of a crRNA is capable of connecting a crRNA to an effector protein. In some embodiments, a single nucleic acid system comprises a guide nucleic acid comprising a crRNA linked to another nucleotide sequence that is capable of being non-covalently bond by an effector protein. In such embodiments, a repeat sequence of a crRNA can be linked to an intermediary sequence. In some embodiments, a single nucleic acid system comprises a guide nucleic acid comprising a crRNA and an intermediary sequence. [319] In some embodiments, a crRNA comprises deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or a combination thereof. In some embodiments, a crRNA comprises about: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 linked nucleotides. In some embodiments, a crRNA comprises at least: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 linked nucleotides. In some embodiments, the length of the crRNA is about 20 to about 120 linked nucleotides. In some embodiments, the length of a crRNA is about 20 to about 100, about 30 to about 100, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 90, about 50 to about 80, about 50 to about 70, or about 50 to about 60 linked 72 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT nucleotides. In some embodiments, the length of a crRNA is about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides. [320] In some embodiments, a crRNA comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the crRNA sequences in TABLE 7. In some embodiments, a crRNA sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the sequences in TABLE 7. In some embodiments, the crRNA sequence comprises a repeat sequence and a spacer sequence. In some embodiments, the repeat sequence is 5’ of the spacer sequence. In some embodiments, a crRNA sequence comprises a repeat sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the sequences recited in TABLE 4, and a spacer sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the sequences recited in TABLE 5. In some embodiments, a crRNA comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 contiguous nucleotides of any one of the crRNA sequences recited in TABLE 7. In some embodiments, a crRNA sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides of any one of the repeat sequences recited in TABLE 4, and at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides of any one of the spacer sequences recited in TABLE 5. sgRNA [321] In some embodiments, a guide nucleic acid comprises an sgRNA. In some embodiments, a guide nucleic acid is an sgRNA. In some embodiments, an sgRNA comprises a first region (FR) and a second region (SR), wherein the FR comprises a handle sequence and the SR comprises a spacer sequence. In some embodiments, the handle sequence and the spacer sequences are directly connected to each other (e.g., covalent bond (phosphodiester bond)). In some embodiments, the handle sequence and the spacer sequence are connected by a linker. [322] In some embodiments, an sgRNA comprises one or more of a handle sequence, an intermediary sequence, a crRNA, a repeat sequence, a spacer sequence, a linker, or a combination thereof. For example, an sgRNA comprises a handle sequence and a spacer sequence; an intermediary sequence and an crRNA; or an intermediary sequence, a repeat sequence and a spacer sequence. [323] In some embodiments, an sgRNA comprises an intermediary sequence and an crRNA. In some embodiments, an intermediary sequence is 5’ to a crRNA in an sgRNA. In some embodiments, an sgRNA comprises a linked intermediary sequence and crRNA. In some embodiments, an intermediary sequence and a crRNA are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester 73 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT bond) In some embodiments, an intermediary sequence and a crRNA are linked in an sgRNA by any suitable linker, examples of which are provided herein. [324] In some embodiments, an sgRNA comprises a handle sequence and a spacer sequence. In some embodiments, a handle sequence is 5’ to a spacer sequence in an sgRNA. In some embodiments, an sgRNA comprises a linked handle sequence and spacer sequence. In some embodiments, a handle sequence and a spacer sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond) In some embodiments, a handle sequence and a spacer sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein. [325] In some embodiments, an sgRNA comprises an intermediary sequence, a repeat sequence, and a spacer sequence. In some embodiments, an intermediary sequence is 5’ to a repeat sequence in an sgRNA. In some embodiments, an sgRNA comprises a linked intermediary sequence and repeat sequence. In some embodiments, an intermediary sequence and a repeat sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond) In some embodiments, an intermediary sequence and a repeat sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein. In some embodiments, a repeat sequence is 5’ to a spacer sequence in an sgRNA. In some embodiments, an sgRNA comprises a linked repeat sequence and spacer sequence. In some embodiments, a repeat sequence and a spacer sequence are linked in an sgRNA directly (e.g, covalently linked, such as through a phosphodiester bond) In some embodiments, a repeat sequence and a spacer sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein. [326] In some embodiments, an sgRNA sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the sequences in TABLE 4, TABLE 5, TABLE 6, TABLE 7, and TABLE 8. In some embodiments, an sgRNA sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the sequences in TABLE 8. In some embodiments, an sgRNA sequence comprises a handle sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the sequences in TABLE 6, and a spacer sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the sequences in TABLE 5. In some embodiments, an sgRNA comprises at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides of any one of the sgRNA sequences recited in TABLE 8. In some embodiments, an sgRNA sequence comprises a handle sequence comprising at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides of any one of the sequences recited in TABLE 6, and a spacer sequence comprising at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides of any one of the sequences recited in TABLE 5. A Dual Nucleic Acid System 74 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [327] In some embodiments, compositions, systems and methods described herein comprise a dual nucleic acid system comprising a crRNA or a nucleotide sequence encoding the crRNA, a tracrRNA or a nucleotide sequence encoding the tracrRNA, and one or more effector protein or a nucleotide sequence encoding the one or more effector protein, wherein the crRNA and the tracrRNA are separate, unlinked molecules, wherein a repeat hybridization region of the tracrRNA is capable of hybridizing with an equal length portion of the crRNA to form a tracrRNA-crRNA duplex, wherein the equal length portion of the crRNA does not include a spacer sequence of the crRNA, and wherein the spacer sequence is capable of hybridizing to a target sequence of the target nucleic acid. In the dual nucleic acid system having a complex of the guide nucleic acid, tracrRNA, and the effector protein, the effector protein is transactivated by the tracrRNA. In other words, activity of effector protein requires binding to a tracrRNA molecule. In some embodiments, the dual nucleic acid system comprises a guide nucleic acid and a tracrRNA, wherein the tracrRNA is an additional nucleic acid capable of at least partially hybridizing to the first region of the guide nucleic acid. In some embodiments, the tracrRNA or additional nucleic acid is capable of at least partially hybridizing to the 5’ end of the second region of the guide nucleic acid. [328] In some embodiments, a repeat hybridization sequence is at the 3’ end of a tracrRNA. In some embodiments, a repeat hybridization sequence comprises a length of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, or about 20 linked nucleotides. In some embodiments, the length of the repeat hybridization sequence is 1 to 20 linked nucleotides. [329] In some embodiments, a tracrRNA and/or tracrRNA-crRNA duplex form a secondary structure that facilitates the binding of an effector protein to a tracrRNA or a tracrRNA-crRNA. In some embodiments, the secondary structure modifies activity of the effector protein on a target nucleic acid. In some embodiments, the secondary structure comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the secondary structure comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). In some embodiments, an effector protein recognizes a secondary structure comprising multiple stem regions. In some embodiments, nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the secondary structure comprises at least two, at least three, at least four, or at least five stem regions. In some embodiments, the secondary structure comprises one or more loops. In some embodiments, the secondary structure comprises at least one, at least two, at least three, at least four, or at least five loops. Engineered Modifications 75 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [330] [Polypeptides (e.g., effector proteins) and nucleic acids (e.g., engineered guide nucleic acids) can be further modified as described herein. Examples are modifications that do not alter the primary sequence of the polypeptides or nucleic acids, such as chemical derivatization of polypeptides (e.g., acylation, acetylation, carboxylation, amidation, etc.), or modifications that do alter the primary sequence of the polypeptide or nucleic acid. Also included are polypeptides that have a modified glycosylation pattern (e.g., those made by: modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes). Also embraced are polypeptides that have phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, or phosphothreonine). [331] Modifications disclosed herein can also include modification of described polypeptides and/or guide nucleic acids through any suitable method, such as molecular biological techniques and/or synthetic chemistry, to improve their resistance to proteolytic degradation, to change the target sequence specificity, to optimize solubility properties, to alter protein activity (e.g., transcription modulatory activity, enzymatic activity, etc.) or to render them more suitable for their intended purpose (e.g., in vivo administration, in vitro methods, or ex vivo applications). Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. In some embodiments, D-amino acids is substituted for some or all of the amino acid residues. Modifications can also include modifications with non-naturally occurring unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, or purity required. [332] Modifications can further include the introduction of various groups to polypeptides and/or guide nucleic acids described herein. For example, groups can be introduced during synthesis or during expression of a polypeptide (e.g., an effector protein), which allow for linking to other molecules or to a surface. Thus, in some embodiments, cysteines are used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, or amino groups for forming amides. [333] Modifications can further include changing of nucleic acids described herein (e.g., engineered guide nucleic acids) to provide the nucleic acid with a new or enhanced feature, such as improved stability. Such modifications of a nucleic acid include a base editing, a base modification, a backbone modification, a sugar modification, or a combination thereof. In some embodiments, the modifications can be of one or more nucleotides, nucleosides, or nucleobases in a nucleic acid. [334] In some embodiments, nucleic acids (e.g., nucleic acids encoding effector proteins, engineered guide nucleic acids, or nucleic acids encoding engineered guide nucleic acids) described herein comprise one or more modifications comprising: 2’O-methyl modified nucleotides (e.g., 2’-O-Methyl (2’OMe) sugar modifications); 2’ fluoro modified nucleotides (e.g., 2’-fluoro (2’-F) sugar modifications); locked nucleic acid (LNA) modified nucleotides; peptide nucleic acid (PNA) modified nucleotides; nucleotides with phosphorothioate linkages; a 5’ cap (e.g., a 7-methylguanylate cap (m7G)), phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other 76 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphor amidates, thionoalkylphosphonates , thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage; phosphorothioate and/or heteroatom internucleoside linkages, such as -CH
2-NH-O-CH
2-, -CH
2- N(CH
3)-O-CH
2- (known as a methylene (methylimino) or MMI backbone), -CH
2-O-N(CH
3)-CH
2-, -CH
2- N(CH
3)- N(CH
3)-CH
2- and -O-N(CH
3)-CH
2-CH
2- (wherein the native phosphodiester internucleotide linkage is represented as -O-P(=O)(OH)-O-CH
2-); morpholino linkages (formed in part from the sugar portion of a nucleoside); morpholino backbones; phosphorodiamidate or other non-phosphodiester internucleoside linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; other backbone modifications having mixed N, O, S and CH
2 component parts; and combinations thereof. Vectors and Multiplexed Expression Vectors [335] Compositions, systems, and methods described herein comprise a vector or a use thereof. A vector can comprise a nucleic acid of interest. In some embodiments, the nucleic acid of interest comprises one or more components of a composition or system described herein. In some embodiments, the nucleic acid of interest comprises a nucleotide sequence that encodes one or more components of the composition or system described herein. In some embodiments, one or more components comprises a polypeptide(s) (e.g., effector protein(s), effector partner(s), fusion protein(s), or a combination thereof), guide nucleic acid(s), target nucleic acid(s), and donor nucleic acid(s). In some embodiments, the component comprises a nucleic acid encoding a polypeptide (e.g., effector protein(s), effector partner(s), fusion protein(s), or a combination thereof), a donor nucleic acid, and a guide nucleic acid or a nucleic acid encoding the guide nucleic acid. In some embodiments, a vector is a part of a vector system. In some embodiments, the vector system comprises a library of vectors each encoding one or more component of a composition or system described herein. In some embodiments, components described herein (e.g., an effector protein, a guide nucleic acid, and/or a donor nucleic acid) are encoded by the same vector. In some embodiments, components described herein (e.g., an effector protein, a guide nucleic acid, and/or a donor nucleic acid) are each encoded by different vectors of the system. In some embodiments, a vector encoding a donor nucleic acid further encodes a target nucleic acid. [336] In some embodiments, a vector comprises a nucleotide sequence encoding one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or a combination thereof as described herein. In some embodiments, the one or more polypeptides comprise at least two polypeptides. In some embodiments, the at least two polypeptides are the same. In some embodiments, the at least two polypeptides are different from each other. In some embodiments, the nucleotide sequence is operably 77 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT linked to a promoter that is operable in a target cell, such as a eukaryotic cell. In some embodiments, the vector comprises the nucleotide sequence encoding 1, 2, 3, 4, or more polypeptides. [337] In some embodiments, a vector encodes one or more of any system components, including but not limited to polypeptides (e.g., effector proteins, effector partners, fusion proteins, or a combination thereof), guide nucleic acids, and target nucleic acids as described herein. In some embodiments, a system component encoding sequence is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell. In some embodiments, a vector encodes 1, 2, 3, 4 or more of any system components. For example, in some embodiments, a vector encodes two or more guide nucleic acids, wherein each guide nucleic acid comprises a different sequence. In some embodiments, a vector encodes the polypeptide and the guide nucleic acid. In some embodiments, a vector encodes the polypeptide, a guide nucleic acid, a donor nucleic acid, or a combination thereof. [338] In some embodiments, a vector comprises one or more guide nucleic acids, or a nucleotide sequence encoding the one or more guide nucleic acids as described herein. In some embodiments, the one or more guide nucleic acids comprise at least two guide nucleic acids. In some embodiments, the at least two guide nucleic acids are the same. In some embodiments, the at least two guide nucleic acids are different from each other. In some embodiments, the guide nucleic acid or the nucleotide sequence encoding the guide nucleic acid is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell. In some embodiments, the vector comprises 1, 2, 3, 4, or more guide nucleic acids. In some embodiments, the vector comprises a nucleotide sequence encoding 1, 2, 3, 4, or more guide nucleic acids. [339] In some embodiments, a vector comprises one or more donor nucleic acids as described herein. In some embodiments, the one or more donor nucleic acids comprise at least two donor nucleic acids. In some embodiments, the at least two donor nucleic acids are the same. In some embodiments, the at least two donor nucleic acids are different from each other. In some embodiments, the vector comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more donor nucleic acids. [340] In some embodiments, a vector comprises or encodes one or more regulatory elements. Regulatory elements, in some embodiments, are referred to as transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, and protein degradation signals, that provide for and/or regulate transcription of a non-coding sequence or a coding sequence and/or regulate translation of an encoded polypeptide. In some embodiments, a vector comprises or encodes for one or more additional elements, such as, for example, replication origins, antibiotic resistance (or a nucleic acid encoding the same), a tag (or a nucleic acid encoding the same), and selectable markers. In some embodiments, a vector comprises or encodes for one or more elements, such as, for example, ribosome binding sites, and RNA splice sites. [341] Vectors described herein can encode a promoter - a regulatory region on a nucleic acid, such as a DNA sequence, capable of initiating transcription of a downstream (3′ direction) coding or non-coding 78 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT sequence. A promoter can be linked at its 3′ terminus to a nucleic acid, the expression or transcription of which is desired, and extends upstream (5′ direction) to include bases or elements necessary to initiate transcription or induce expression, which could be measured at a detectable level. A promoter can comprise a nucleotide sequence, referred to herein as a “promoter sequence”. The promoter sequence can include a transcription initiation site, and one or more protein binding domains responsible for the binding of transcription machinery, such as RNA polymerase. When eukaryotic promoters are used, such promoters can contain “TATA” boxes and “CAT” boxes. In some embodiments, various promoters, including inducible promoters, are used to drive expression, i.e., transcriptional activation, of the nucleic acid of interest. Accordingly, in some embodiments, the nucleic acid of interest can be operably linked to a promoter. [342] In some embodiments, promotors comprises any suitable type of promoter envisioned for the compositions, systems, and methods described herein. Examples include constitutively active promoters (e.g., CMV promoter), inducible promoters (e.g., heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.), spatially restricted and/or temporally restricted promoters (e.g., a tissue specific promoter, a cell type specific promoter, a site-specific promoter, etc.), etc. Suitable promoters include, but are not limited to: SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6), an enhanced U6 promoter, and a human Hl promoter (Hl). By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by 2 fold, 5 fold, 10 fold, 50 fold, by 100 fold, 500 fold, or by 1000 fold, or more. In addition, vectors used for providing a nucleic acid that, when transcribed, produces a guide nucleic acid and/or a nucleic acid that encodes a polypeptide (e.g., an effector protein, an effector partner, a fusion protein, or a combination thereof) to a cell comprising nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the guide nucleic acid and/or the polypeptide. [343] In general, vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein. In some embodiments, the vector comprises a nucleotide sequence of a promoter. In some embodiments, the vector comprises two promoters. In some embodiments, the vector comprises three promoters. In some embodiments, a length of the promoter is less than about 500, less than about 400, less than about 300, or less than about 200 linked nucleotides. In some embodiments, a length of the promoter is at least 100, at least 200, at least 300, at least 400, or at least 500 linked nucleotides. Non-limiting examples of promoters include CMV, 7SK, EF1a, RPBSA, hPGK, EFS, SV40, PGK1, Ubc, human beta actin, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GAL1-10, H1, TEF1, GDS, ADH1, CaMV35S, HSV TK, Ubi, U6, MNDU3, MSCV, MND and CAG. 79 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [344] In some embodiments, some promoters (e.g., U6, enhanced U6, Hl and 7SK) prefers the nucleic acid being transcribed having “g” nucleotide at the 5’ end of the coding sequence. Accordingly, when such coding sequence is expressed, it comprises an additional “g” nucleotide at 5’ end. In some embodiments, vectors provided herein comprise a promotor driving expression or transcription of any one of the guide nucleic acids described herein (e.g., TABLE 4, TABLE 5, TABLE 6, TABLE 7, and TABLE 8) further comprises “g” nucleotide at 5’ end of the guide nucleic acid, wherein the promotor is selected from U6, enhanced U6, Hl and 7SK. [345] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter only drives expression of its corresponding coding sequence (e.g., polypeptide or guide nucleic acid) when a signal is present, e.g., a hormone, a small molecule, a peptide. Non-limiting examples of inducible promoters are the T7 RNA polymerase promoter, the T3 RNA polymerase promoter, the Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, a tetracycline-regulated promoter (tetracycline-inducible or tetracycline-repressible), a steroid regulated promoter, a metal- regulated promoter, and an estrogen receptor-regulated promoter. In some embodiments, the promoter is an activation-inducible promoter, such as a CD69 promoter as described further in Kulemzin et al., (2019), BMC Med Genomics, 12:44. In some embodiments, the promoter for expressing effector protein is a muscle-specific promoter. In some embodiments, the muscle-specific promoter comprises Ck8e, SPC5-12, or Desmin promoter sequence. Non-limiting examples of muscle-specific promoters include HAS and MCK. In some embodiments, the promoter for expressing a polypeptide (e.g., an effector protein, an effector partner, a fusion protein, or a combination thereof) is a ubiquitous promoter. In some embodiments, the ubiquitous promoter comprises MND or CAG promoter sequence. [346] In some embodiments, the promoters are prokaryotic promoters (e.g., drive expression of a gene in a prokaryotic cell). In some embodiments, the promoters are eukaryotic promoters, (e.g., drive expression of a gene in a eukaryotic cell). In some embodiments, the promoter is EF1a. In some embodiments, the promoter is ubiquitin. In some embodiments, vectors are bicistronic or polycistronic vector (e.g., having or involving two or more loci responsible for generating a protein) having an internal ribosome entry site (IRES) is for translation initiation in a cap-independent manner. [347] In some embodiments, a vector described herein is a nucleic acid expression vector. In some embodiments, a vector described herein is a recombinant expression vector. In some embodiments, a vector described herein is a messenger RNA. In some embodiments, a vector comprising the recombinant nucleic acid as described herein, wherein the vector is a viral vector, an adeno associated viral (AAV) vector, a retroviral vector, or a lentiviral vector. In some embodiments, a vector described herein or a recombinant nucleic acid described herein is comprised in a cell. In some embodiments, a recombinant nucleic acid integrated into a genomic DNA sequence of the cell, wherein the cell is a eukaryotic cell or a prokaryotic cell. 80 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [348] In some embodiments, a vector described herein is a delivery vector. In some embodiments, the delivery vector is a eukaryotic vector, a prokaryotic vector (e.g., a bacterial vector) a viral vector, or a combination thereof. In some embodiments, the delivery vehicle is a non-viral vector. In some embodiments, the delivery vector is a plasmid. In some embodiments, the plasmid comprises DNA. In some embodiments, the plasmid comprises RNA. In some embodiments, the plasmid comprises circular double- stranded DNA. In some embodiments, the plasmid is linear. In some embodiments, the plasmid comprises one or more coding sequences of interest and one or more regulatory elements. In some embodiments, the plasmid comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria. In some embodiments, the plasmid is a minicircle plasmid. In some embodiments, the plasmid contains one or more genes that provide a selective marker to induce a target cell to retain the plasmid. In some examples, the plasmid is formulated for delivery through injection by a needle carrying syringe. In some examples, the plasmid is formulated for delivery via electroporation. In some examples, the plasmids are engineered through synthetic or other suitable means known in the art. For example, in some embodiments, the genetic elements are assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which is then be readily ligated to another genetic sequence. [349] In some embodiments, vectors comprise an enhancer. Enhancers are nucleotide sequences that have the effect of enhancing promoter activity. In some embodiments, enhancers augment transcription regardless of the orientation of their sequence. In some embodiments, enhancers activate transcription from a distance of several kilo basepairs. Furthermore, enhancers are located optionally upstream or downstream of a gene region to be transcribed, and/or located within the gene, to activate the transcription. Exemplary enhancers include, but are not limited to, WPRE; CMV enhancers; the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol.78(3), p.1527-31, 1981); and the genome region of human growth hormone (J Immunol., Vol.155(3), p.1286-95, 1995). [350] In some embodiments, a vector (e.g., nucleic acid expression vector) described herein encodes a guide nucleic acid that comprises: a spacer sequence that is at least 90% identical to any one of the sequences recited in TABLE 5, or a sgRNA that is at least 80% identical to any one of the sequences recited in TABLE 8, wherein the vector is a viral vector. In some embodiments, the viral vector is an adeno associated viral (AAV) vector. In some embodiments, the viral vector comprises a nucleotide sequence of a first promoter, wherein the first promoter drives transcription of a nucleotide sequence encoding the guide nucleic acid, and wherein the first promoter is selected from a group consisting of CMV, EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GAL1-10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, CaMV35S, SV40, CMV, 7SK, and HSV TK. In some embodiments, the vector comprises a nucleic acid sequence encoding a polypeptide that comprises an amino acid sequence that has at least 80% identity to the sequence recited in TABLE 1 or a variant thereof. In some embodiments, the vector comprises a nucleotide sequence of a second promoter, wherein the second 81 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT promoter drives expression of the polypeptide, and wherein the second promoter is a ubiquitous promoter or a site-specific promoter. In some embodiments, the ubiquitous promoter is selected from a group consisting of MND and CAG. In some embodiments, the site-specific promoter is selected from a group consisting of Ck8e, Spc5-12, and Desmin. In some embodiments, the vector comprises an enhancer, wherein the enhancer is a nucleotide sequence having the effect of enhancing promoter activity, wherein the enhancer is selected from a group consisting of WPRE enhancer, CMV enhancers, the R-U5′ segment in LTR of HTLV-I, SV40 enhancer, the intron sequence between exons 2 and 3 of rabbit β-globin, and the genome region of human growth hormone. In some embodiments, the vector comprises a poly A signal sequence. In some embodiments, the vector comprises a nucleotide sequence encoding a first guide nucleic acid and a nucleotide sequence encoding a second guide nucleic acid, and wherein the first guide nucleic acid is different from the second guide nucleic acid. In some embodiments, the vector comprises a nucleotide sequence of a third promoter, wherein the third promoter drives transcription of a nucleotide sequence encoding the second guide nucleic acid, wherein the third promoter is selected from a group consisting of CMV, EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GAL1-10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, CaMV35S, SV40, CMV, 7SK, and HSV TK, and wherein the first promoter and the third promoter are different. Administration of a Non-Viral Vector [351] In some embodiments, an administration of a non-viral vector comprises contacting a cell, such as a host cell, with the non-viral vector. In some embodiments, a physical method or a chemical method is employed for delivering the vector into the cell. Exemplary physical methods include electroporation, gene gun, sonoporation, magnetofection, or hydrodynamic delivery. Exemplary chemical methods include delivery of the recombinant polynucleotide by liposomes such as, cationic lipids or neutral lipids; lipofection; dendrimers; lipid nanoparticle (LNP); or cell-penetrating peptides. [352] In some embodiments, a vector is administered as part of a method of nucleic acid detection, editing, and/or treatment as described herein. In some embodiments, a vector is administered in a single vehicle, such as a single expression vector. In some embodiments, at least two of the three components, a nucleic acid encoding one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or a combination thereof), one or more donor nucleic acids, and one or more guide nucleic acids or a nucleic acid encoding the one or more guide nucleic acid, are provided in the single expression vector. In some embodiments, components, such as a guide nucleic acid and a polypeptide (e.g., effector protein, effector partner, fusion protein, or a combination thereof), are encoded by the same vector. In some embodiments, a polypeptide (e.g., effector protein, effector partner, fusion protein, or a combination thereof) (or a nucleic acid encoding same) and/or an engineered guide nucleic acid (or a nucleic acid that, when transcribed, produces same) are not co-administered with donor nucleic acid in a single vehicle. In some embodiments, a polypeptides (e.g., effector protein, effector partner, fusion protein, or a combination thereof) (or a nucleic acid encoding same), an engineered guide nucleic acid (or a nucleic acid that, when transcribed, produces 82 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT same), and/or donor nucleic acid are administered in one or more or two or more vehicles, such as one or more, or two or more expression vectors. [353] In some embodiments, a vector system is administered as part of a method of nucleic acid detection, editing, and/or treatment as described herein, wherein at least two vectors are co-administered. In some embodiments, the at least two vectors comprise different components. In some embodiments, the at least two vectors comprise the same component having different sequences. In some embodiments, at least one of the three components, a nucleic acid encoding one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or a combination thereof), one or more donor nucleic acids, and one or more guide nucleic acids or a nucleic acid encoding the one or more guide nucleic acids, or a variant thereof is provided in a different vector. In some embodiments, the nucleic acid encoding the polypeptides (e.g., effector proteins, effector partners, fusion proteins, or a combination thereof), and a guide nucleic acid or a nucleic acid encoding the guide nucleic acid are provided in different vectors. In some embodiments, the donor nucleic acid is encoded by a different vector than the vector encoding the effector protein and the guide nucleic acid. Lipid Particles and Non-Viral Vectors [354] In some embodiments, compositions and systems provided herein comprise a lipid or a lipid particle. In some embodiments, a lipid particle is a lipid nanoparticle (LNP). In some embodiments, a lipid or a lipid nanoparticle can encapsulate a nucleic acid (e.g., DNA or RNA) encoding one or more of the components as described herein. In some embodiments, a lipid or a lipid nanoparticle can encapsulate an expression vector as described herein. LNPs are a non-viral delivery system for delivery of the composition and/or system components described herein. LNPs are particularly effective for delivery of nucleic acids. Beneficial properties of LNP include ease of manufacture, low cytotoxicity and immunogenicity, high efficiency of nucleic acid encapsulation and cell transfection, multi-dosing capabilities and flexibility of design (Kulkarni et al., (2018) Nucleic Acid Therapeutics, 28(3):146-157). In some embodiments, compositions and methods comprise a lipid, polymer, nanoparticle, or a combination thereof, or use thereof, to introduce one or more effector proteins, one or more guide nucleic acids, one or more donor nucleic acids, or a combinations thereof to a cell. Non-limiting examples of lipids and polymers are cationic polymers, cationic lipids, ionizable lipids, or bio-responsive polymers. In some embodiments, the ionizable lipids exploits chemical-physical properties of the endosomal environment (e.g., pH) offering improved delivery of nucleic acids. In some embodiments, the ionizable lipids are neutral at physiological pH. In some embodiments, the ionizable lipids are protonated under acidic pH. In some embodiments, the bio- responsive polymer exploits chemical-physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space. [355] In some embodiments, a LNP comprises an outer shell and an inner core. In some embodiments, the outer shell comprises lipids. In some embodiments, the lipids comprise modified lipids. In some embodiments, the modified lipids comprise pegylated lipids. In some embodiments, the lipids comprise one 83 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT or more of cationic lipids, anionic lipids, ionizable lipids, and non-ionic lipids. In some embodiments, the LNP comprises one or more of 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l-palmitoyl-2- oleoylsn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), 1,2-dimyristoyl-sn-glycerol, and methoxypolyethylene glycol (DMG-PEChooo), derivatives, analogs, or variants thereof or any combination of the foregoing. [356] In some embodiments, the LNP comprises one or more ionizable lipid. Such ionizable lipids include, but are not limited to: 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien- 1-yl-10,13-nonadecadien-1-yl ester (DLin-MC3-DMA, CAS No. 1224606-06-7); N,N-dimethyl-2,2-di- (9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethanamine (DLin-KC2-DMA, CAS No. 1190197-97- 7); 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102, CAS No. 2089251-47-6); 8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]-octanoic acid, 1- octylnonyl ester (Lipid 5, CAS No. 2089251-33-0); 1,1′-[[2-[4-[2-[[2-[bis(2- hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2- dodecanol (C12-200, CAS No. 1220890-25-4); 2-hexyl-decanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di- 6,1-hexanediyl] ester (ALC-0315, CAS No. 2036272-55-4); 9,12-octadecadienoic acid, (9Z,12Z)- 1,1′,1′′,1′′′-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1-butanediylnitrilodi-4,1-butanediyl)] ester (OF-C4-Deg- Lin, CAS No. 1853203-01-6); bis(2-(dodecyldisulfaneyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14- dithia-3,6-diazahexacosyl)azanediyl)dipropionate (BAMEA-O16B, CAS No. 2490668-30-7); 3,6-bis[4- [bis[(9Z,12Z)-2-hydroxy-9,12-octadecadien-1-yl]amino]butyl]-2,5-piperazinedione (OF-02, CAS No. 1883431-67-1); tetrakis(8-methylnonyl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1- diyl))bis(azanetriyl))tetrapropionate (306Oi10, CAS No. 2322290-93-5); tetrakis(2- (octyldisulfaneyl)ethyl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1- diyl))bis(azanetriyl))tetrapropionate (306-O12B, CAS No. 2566523-06-4); bis(2-butyloctyl) 10-(N-(3- (dimethylamino)propyl)nonanamido)nonadecanedioate (Lipid A9, CAS No. 2036272-50-9); Arcturus Lipid 2,2 (8,8) 4C CH3 (ATX-0114, CAS No. 2230647-28-4) ); di((Z)-non-2-en-1-yl) 8,8'-((2-((2- (dimethylamino)ethyl)thio)acetyl)azanediyl)dioctanoate (ATX-001, CAS No. 1777792-33-2); di((Z)-non- 2-en-1-yl) 8,8'-((((2-(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX-002, CAS No. 1777792-34-3); Genevant CL1 (CAS No. 1450888-71-7); LP01; hexa(octan-3-yl) 9,9',9",9"',9"",9"'"- ((((benzene-l,3,5-tricarbonyl)yris(azanediyl)) tris (propane-3,1-diyl)) tris(azanetriyl))hexanonanoate (FTT5); 5A2-SC8 (CAS No. 1857341-90-2); COATSOME® SS-OP; derivatives; analogs; or variants thereof. In some embodiments, the LNP comprise a combination of two, three, four, five or more of the foregoing ionizable lipids. [357] In some embodiments, the LNP has a negative net overall charge prior to complexation with one or more of a guide nucleic acid, a nucleic acid encoding the one or more guide nucleic acid, a nucleic acid encoding a polypeptide (e.g., effector protein, effector partner, fusion protein, or a combination thereof), and/or a donor nucleic acid. In some embodiments, the inner core is a hydrophobic core. In some embodiments, the one or more of a guide nucleic acid, the nucleic acid encoding the one or more guide 84 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT nucleic acid, the nucleic acid encoding the polypeptide, and/or the donor nucleic acid forms a complex with one or more of the cationic lipids and the ionizable lipids. In some embodiments, the nucleic acid encoding the polypeptide or the nucleic acid encoding the guide nucleic acid is self-replicating. [358] In some embodiments, a LNP comprises one or more of cationic lipids, ionizable lipids, and modified versions thereof. In some embodiments, the ionizable lipid comprises N1,N3,N5-tris(3- (didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3) or a derivative thereof. Accordingly, in some embodiments, the LNP comprises one or more of TT3 and pegylated TT3. The publication WO2016187531 is hereby incorporated by reference in its entirety, which describes representative LNP formulations in Table 2 and Table 3, and representative methods of delivering LNP formulations in Example 7. [359] In some embodiments, a LNP comprises a lipid composition targeting to a specific organ. In some embodiments, the lipid composition comprises lipids having a specific alkyl chain length that controls accumulation of the LNP in the specific organ (e.g., liver or spleen). In some embodiments, the lipid composition comprises a biomimetic lipid that controls accumulation of the LNP in the specific organ (e.g., brain). In some embodiments, the lipid composition comprises lipid derivatives (e.g., cholesterol derivatives) that controls accumulation of the LNP in a specific cell (e.g., liver endothelial cells, Kupffer cells, hepatocytes). [360] In some embodiments, the LNP described herein comprises nucleic acids (e.g., DNA or RNA) encoding an effector protein described herein, an effector partner described herein, a fusion protein described herein, a guide nucleic acid described herein, or a combination thereof. In some embodiments, the LNP comprises an mRNA that produces an effector protein described herein, an effector partner described herein, or a fusion protein described herein when translated. In some embodiments, the LNP comprises chemically modified guide nucleic acids. Delivery of Viral Vectors [361] In some embodiments, a vector described herein comprises a viral vector. In some embodiments, the viral vector comprises a nucleic acid to be delivered into a host cell by a recombinantly produced virus or viral particle. In some embodiments, the nucleic acid comprises single-stranded or double stranded, linear or circular, segmented or non-segmented. In some embodiments, the nucleic acid comprises DNA, RNA, or a combination thereof. In some embodiments, the vector is an adeno-associated viral vector. There are a variety of viral vectors that are associated with various types of viruses, including but not limited to retroviruses (e.g., lentiviruses and γ-retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno- associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses. In some embodiments, the vector is an adeno-associated viral (AAV) vector. In some embodiments, the viral vector is a recombinant viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the retroviral vector comprises gamma- retroviral vector. In some embodiments, a viral vector provided herein is derived from or based on any such virus. For example, in some embodiments, the gamma-retroviral vector is derived from a Moloney Murine 85 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or a Murine Stem cell Virus (MSCV) genome. In some embodiments, the lentiviral vector is derived from the human immunodeficiency virus (HIV) genome. In some embodiments, the viral vector is a chimeric viral vector. In some embodiments, the chimeric viral vector comprises viral portions from two or more viruses. In some embodiments, the viral vector corresponds to a virus of a specific serotype. [362] In some embodiments, a viral vector is an adeno-associated viral vector (AAV vector). In some embodiments, a viral particle that delivers a viral vector described herein is an AAV. In some embodiments, the AAV comprises any AAV known in the art. In some embodiments, the viral vector corresponds to a virus of a specific AAV serotype. In some embodiments, the AAV serotype is selected from an AAV1 serotype, an AAV2 serotype, AAV3 serotype, an AAV4 serotype, AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, an AAV9 serotype, an AAV10 serotype, an AAV11 serotype, an AAV12 serotype, an AAV-rh10 serotype, and any combination, derivative, or variant thereof. In some embodiments, the AAV vector is a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV, or a combination thereof. scAAV genomes are generally known in the art and contain both DNA strands which can anneal together to form double-stranded DNA. [363] In some embodiments, an AAV vector described herein is a chimeric AAV vector. In some embodiments, the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes. In some embodiments, a chimeric AAV vector is genetically engineered to increase transduction efficiency, selectivity, or a combination thereof. [364] In some embodiments, AAV vector described herein comprises two inverted terminal repeats (ITRs). According, in some embodiments, the viral vector provided herein comprises two inverted terminal repeats of AAV. A nucleotide sequence between the ITRs of an AAV vector provided herein comprises a sequence encoding genome editing tools. In some embodiments, the genome editing tools comprise a nucleic acid encoding one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or a combination thereof), a nucleic acid encoding the one or more polypeptides comprising a heterologous peptide (e.g., a nuclear localization signal (NLS), polyA tail), one or more guide nucleic acids, a nucleic acid encoding the one or more guide nucleic acids, respective promoter(s), one or more donor nucleic acid, or a combination thereof. In some embodiments, viral vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein. In some embodiments, a coding region of the AAV vector forms an intramolecular double-stranded DNA template thereby generating the AAV vector that is a self-complementary AAV (scAAV) vector. In some embodiments, the scAAV vector comprises the sequence encoding genome editing tools that has a length of about 2 kb to about 3 kb. In some embodiments, the AAV vector provided herein is a self-inactivating AAV vector. In some embodiments, the AAV vector provided herein comprises a modification, such as an insertion, deletion, chemical alteration, or synthetic modification, relative to a wild-type AAV vector. 86 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT Producing AAV Delivery Vectors [365] In some embodiments, methods of producing AAV delivery vectors herein comprise packaging a nucleic acid encoding a polypeptide (e.g., effector protein, effector partner, fusion protein, or a combination thereof) and a guide nucleic acid, or a combination thereof, into an AAV vector. In some embodiments, methods of producing the delivery vector comprises, (a) contacting a cell with at least one nucleic acid encoding: (i) a guide nucleic acid; (ii) a Replication (Rep) gene; and (iii) a Capsid (Cap) gene that encodes an AAV capsid protein; (b) expressing the AAV capsid protein in the cell; (c) assembling an AAV particle; and (d) packaging the polypeptide encoding nucleic acid into the AAV particle, thereby generating an AAV delivery vector. In some embodiments, promoters, stuffer sequences, and any combination thereof are packaged in the AAV vector. In some embodiments, the AAV vector is package 1, 2, 3, 4, or 5 guide nucleic acids or copies thereof. In some embodiments, the AAV vector comprises inverted terminal repeats, e.g., a 5’ inverted terminal repeat and a 3’ inverted terminal repeat. In some embodiments, the AAV vector comprises a mutated inverted terminal repeat that lacks a terminal resolution site. [366] In some embodiments, a hybrid AAV vector is produced by transcapsidation, e.g., packaging an inverted terminal repeat (ITR) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes are not the same. In some examples, the Rep gene and ITR from a first AAV serotype (e.g., AAV2) is used in a capsid from a second AAV serotype (e.g., AAV9), wherein the first and second AAV serotypes are not the same. As a non-limiting example, a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein is indicated AAV2/9. In some examples, the hybrid AAV delivery vector comprises an AAV2/1, AAV2/2, AAV 2/4, AAV2/5, AAV2/8, or AAV2/9 vector. Producing AAV Particles [367] In some embodiments, AAV particles described herein are recombinant AAV (rAAV). In some embodiments, rAAV particles are generated by transfecting AAV producing cells with an AAV-containing plasmid carrying the sequence encoding the genome editing tools, a plasmid that carries viral encoding regions, i.e., Rep and Cap gene regions; and a plasmid that provides the helper genes such as E1A, E1B, E2A, E4ORF6 and VA. In some embodiments, the AAV producing cells are mammalian cells. In some embodiments, host cells for rAAV viral particle production are mammalian cells. In some embodiments, a mammalian cell for rAAV viral particle production is a COS cell, a HEK293T cell, a HeLa cell, a KB cell, a variant thereof, or a combination thereof. In some embodiments, rAAV virus particles can be produced in the mammalian cell culture system by providing the rAAV plasmid to the mammalian cell. In some embodiments, producing rAAV virus particles in a mammalian cell comprises transfecting vectors that express the rep protein, the capsid protein, and the gene-of-interest expression construct flanked by the ITR sequence on the 5’ and 3’ ends. Methods of such processes are provided in, for example, Naso et al., BioDrugs, 2017 Aug;31(4):317-334 and Benskey et al., (2019), Methods Mol Biol., 1937:3-26, each of which is incorporated by reference in their entireties. 87 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [368] In some embodiments, rAAV is produced in a non-mammalian cell. In some embodiments, rAAV is produced in an insect cell. In some embodiments, the insect cell for producing rAAV viral particles comprises a Sf9 cell. In some embodiments, production of rAAV virus particles in insect cells comprises infecting the insect cells with baculovirus. In some embodiments, production of rAAV virus particles in insect cells comprises infecting the insect cells with three recombinant baculoviruses, one carrying the cap gene, one carrying the rep gene, and one carrying the gene-of-interest expression construct enclosed by an ITR on both the 5’ and 3’ end. In some embodiments, rAAV virus particles are produced by the One Bac system. In some embodiments, rAAV virus particles can be produced by the Two Bac system. In some embodiments, in the Two Bac system, the rep gene and the cap gene of the AAV is integrated into one baculovirus virus genome, and the ITR sequence and the gene-of-interest expression construct is integrated into another baculovirus virus genome. In some embodiments, in the One Bac system, an insect cell line that expresses both the rep protein and the capsid protein is established and infected with a baculovirus virus integrated with the ITR sequence and the gene-of-interest expression construct. Details of such processes are provided in, for example, Smith et. al., (1983), Mol. Cell. Biol., 3(12):2156-65; Urabe et al., (2002), Hum. Gene. Ther., 1;13(16):1935-43; and Benskey et al., (2019), Methods Mol Biol., 1937:3-26, each of which is incorporated by reference in its entirety. Target Nucleic Acids [369] Disclosed herein are compositions, systems and methods for detecting and/or editing a target nucleic acid. In some embodiments, the target nucleic acid is a double stranded nucleic acid. In some embodiments, the target nucleic acid is a single stranded nucleic acid. Alternatively, or in combination, the target nucleic acid is a double stranded nucleic acid and is prepared into single stranded nucleic acids before or upon contacting an RNP. In some embodiments, the single stranded nucleic acid comprises a RNA, wherein the RNA comprises a mRNA, a rRNA, a tRNA, a non-coding RNA, a long non-coding RNA, a microRNA (miRNA), and a single-stranded RNA (ssRNA). In some embodiments, the target nucleic acid is complementary DNA (cDNA) synthesized from a single-stranded RNA template in a reaction catalyzed by a reverse transcriptase. In some embodiments, the target nucleic acid comprises an RNA, a DNA, or a combination thereof. In some embodiments, guide nucleic acids described herein hybridize to a portion of the target nucleic acid. In some embodiments, the target nucleic acid is from a virus, a parasite, or a bacterium described herein. [370] In some embodiments, a target nucleic acid comprising a target sequence comprises a PAM sequence. In some embodiments, the PAM sequence is adjacent to the target sequence. In some embodiments, the PAM sequence adjacent to a target sequence of a target nucleic acid. In some embodiments, the PAM sequence is 3’ to the target sequence. In some embodiments, the PAM sequence is directly 3’ to the target sequence. In some embodiments, the PAM sequence 5’ to the target sequence. In some embodiments, the PAM sequence is directly 5’ to the target sequence. In some embodiments, the target nucleic acid as described in the methods herein does not initially comprise a PAM sequence. However, any target nucleic acid of interest that is generated using the methods described herein to 88 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT comprise a PAM sequence, and thus be a PAM target nucleic acid. A PAM target nucleic acid, as used herein, refers to a target nucleic acid that has been amplified to insert a PAM sequence that is recognized by a polypeptide system. [371] In some embodiments, a target nucleic acid comprises 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 linked nucleotides. In some embodiments, the target nucleic acid comprises 10 to 90, 20 to 80, 30 to 70, or 40 to 60 linked nucleotides. In some embodiments, the target nucleic acid comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 linked nucleotides. In some embodiments, the target nucleic acid comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 linked nucleotides. In some embodiments, the target sequence in the target nucleic acid comprises at least 10 contiguous nucleotides that are complementary to the guide nucleic acid or engineered guide nucleic acid. [372] In some embodiments, compositions, systems, and methods described herein comprise a target nucleic acid that is responsible for a disease, contain a mutation (e.g., single strand polymorphism, point mutation, insertion, or deletion), be contained in an amplicon, or be uniquely identifiable from the surrounding nucleic acids (e.g., contain a unique sequence of nucleotides). In some embodiments, the target nucleic acid has undergone a modification (e.g., an editing) after contacting with an RNP. In some embodiments, the editing is a change in the sequence of the target nucleic acid. In some embodiments, the change comprises an insertion, deletion, or substitution of one or more nucleotides compared to the target nucleic acid that has not undergone any modification. In some embodiments, the disease can comprise, at least in part, an inherited disorder, a neurological disorder, or both. In some embodiments, the neurological disorder is a neuromuscular disorder. In some embodiments, the neuromuscular disorder comprises: muscular dystrophy (MD); myotonic dystrophy (DM); myotonic dystrophy type 1 (DM1); dystrophia myotonica; myotonia atrophica; myotonia dystrophica; Steinert disease; Curschmann–Batten–Steinert syndrome; hypotonia; cardiomyopathy; MD-associated cardiomyopathy. In some embodiments, the disease is any one of the diseases listed in TABLE 10. In some embodiments, a target nucleic acid comprises a portion or a specific region of a nucleic acid from a genomic locus, any DNA amplicon of, a reverse transcribed mRNA, or a cDNA from a gene described herein. In some embodiments, the target nucleic acid is an amplicon of at least a portion of a gene. Non-limiting examples of genes are recited in TABLE 9. Nucleic acid sequences of target nucleic acids and/or corresponding genes are readily available in public databases as known and used in the art. In some embodiments, the target nucleic acid is selected from TABLE 9. In some embodiments, the target nucleic acid comprises one or more target sequences. In some embodiments, the one or more target sequence is within any one of the target nucleic acids recited in TABLE 9. In some embodiments, the target nucleic acid is any one of the target nucleic acids listed in TABLE 9 or 9.1. In some embodiments, the target nucleic acid is the DMPK gene. In some embodiments, the target nucleic acid modification occurs in any one of the locations listed in TABLE 9.2. 89 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [373] Myotonic dystrophy (DM) is an autosomal, dominantly inherited, neuromuscular disorder which affects at least 1 in 8,000 people, though it is more prevalent in people of European ancestry. DM is a genetic cause of progressive neuromuscular degeneration affecting tissues such as eye tissue, cardiac muscle, skeletal muscle, smooth muscle, nervous tissue, and neurons. DM is categorized into DM type 1 (DM1) and DM type 2 (DM2) , with DM1 being the most common. While DM1 is caused by expansion of an expanded unstable CTG repeat in the 3′ untranslated region of the DMPK gene (DM Protein Kinase gene; Chromosome 19: NCBI Reference Sequence: NC_000019.10), and DM2 is caused by an expanded CCTG repeat in intron 1 of the CNBP gene (Cellular Nucleic Acid Binding Protein gene), both DM1 and DM2 lead to the expression of dominant-acting RNAs, which lead to RNA toxicity within a cell. Currently there are no disease-modifying treatments for either disorder. However, therapeutic treatments directed at gene silencing or silencing transcription may alleviate RNA toxicity related to repeat expansion mutations. [374] Clinical expression of DM1 is variable, presenting a progressive neuromuscular degeneration that affects distal muscles more than proximal. Symptoms include muscle weakness, myotonia (reduced or impaired muscle relaxation), intellectual disability, CNS impairment and/or degeneration, early cataracts, endocrine irregularities, cardiac arrhythmias, cardiomyopathy, testicular atrophy, insulin resistance, and changes in neuropsychological function. People with DM1 have 50 to 5,000 CTG repeats in the noncoding region of the DMPK gene in most cells. However, the number of repeats may be even greater in certain types of cells, such as muscle cells. Since the autosomal-dominant CTG repeat expansion causing DM1 is present from conception, the pathophysiology of the disease may manifest at birth (congenital) or early life (childhood onset). Furthermore, pathogenic alleles, which are inherited in an autosomal dominant manner, may expand in length during gametogenesis, resulting in the transmission of longer trinucleotide repeat alleles, which may lead to an earlier onset and/or a more severe pathophysiology as compared to the parent. Reference ranges for allele sizes were established by the Second International Myotonic Dystrophy Consortium (IDMC) (1999). Clinical expression of DM1, which may span from mild to severe, is categorized into three phenotypes: (i) mild (50-150 CTG repeats, age of onset 20-70 years, normal life span, clinical signs include cataracts and mild myotonia (sustained muscle contraction)), (ii) classic (100-1,000 CTG repeats, age of onset 10-30 years, reduced life span, clinical signs include cataracts, myotonia, muscle weakness, cardiac arrhythmia), and (iii) congenital (>1,000 CTG repeats, age of onset birth-10 years, greatly reduced life span, clinical signs include infantile hypotonia, respiratory deficit, intellectual disability, muscular degeneration, carcinoma). [375] Overall, the accumulation of DMPK transcripts (mutant DMPK mRNA) in nuclear foci and/or decreased DMPK protein levels may contribute to the pathology of DM1. The pathogenic mechanisms that contribute to the pathophysiology of DM1 are generally believed to be related in part to DMPK mRNA- induced toxicity as well as toxic RNA-induced splicing misregulation. For example, mutant DMPK mRNA interferes with muscle-blind–like proteins (MBNL) in alternate splicing modulation, resulting in widespread splicing abnormalities. Since the autosomal-dominant CTG repeat expansion in DMPK is present from conception, developmentally regulated splicing pattern changes may not occur, resulting in 90 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT adult DM1 tissues (e.g., CNS tissue and/or skeletal muscle tissue) comprising embryonic and/or fetal splicing patterns. Thus, the misregulated RNA metabolism in patients’ tissues may lead to the expression of normal mRNA variants during inappropriate developmental stages and/or in inappropriate tissues. Additionally, changes in the expression of the DMPK protein, such as abnormally low levels of DMPK protein expression and/or abnormal developmental patterns of DMPK protein expression, further contribute to the pathophysiology of DM1. The DMPK protein is a Ser/Thr protein kinase homologous to the p21- activated kinases MRCK and ROCK/rho-kinase/ROK. The most abundant isoform of DMPK is an 80 kDa protein mainly expressed in smooth, skeletal and cardiac muscles. Functionally, the DMPK protein has been shown to influence intracellular trafficking (e.g., Ca2+ cycling and ion-channel gating) as well as maintaining cytoarchitecture of skeletal and smooth muscles. DMPK transcripts (mutant DMPK mRNA) in nuclear foci and/or decreased DMPK protein levels may contribute to the pathology of DM1. The pathogenic mechanisms that contribute to the pathophysiology of DM1 are generally believed to be related in part to DMPK mRNA. In some embodiments, DMPK-associated nucleic acids from any vertebrate source (e.g., mammals, primates, humans, dogs, rodents, mice, rats) can be interchangeably referred to herein as DMPK, DMPK gene, DMPK transcript, DMPK mRNA, and DM1. [376] The human DMPK gene contains 15 exons and spans ∼70 kd, and is located on chromosome 19, at cytogenetic location 19q13.32. An exemplary amino acid sequence of DMPK, UniProtKB protein Q09013 (DMPK_HUMAN), is in TABLE 9.1 as SEQ ID NO: 66. An exemplary encoding nucleic acid sequence of human DMPK can be found at NCBI Reference Sequence: NM_004409.5 and is provided in TABLE 9.1. The genomic locations of DMPK can be found at Ensembl No. ENST00000291270.9 Human (GRCh38/hg38) and is provided, at least in part, in TABLE 9.1 as SEQ ID NO: 67. In some embodiments, at least partial sequences of certain exemplary exonic and intronic genomic locations can be found in TABLE 9.2 as SEQ ID NOS: 68-98. [377] In some embodiments, the target sequence is within the human DMPK gene. In some embodiments, the target sequence is within a UTR sequence containing repeats of (CTG)
n of the human DMPK gene. In some embodiments, the target sequence is downstream of the coding region of exon 14 of the human DMPK gene. In some embodiments, the target sequence is within a UTR sequence containing repeats of (CTG)
n downstream of the coding region of exon 14 of the human DMPK gene. In some embodiments, the target sequence is located within about 1 to about 300 nucleotides, about 10 to about 250, about 20 to about 200, about 30 to about 150, about 40 to about 100, or about 50 nucleotides 5’ of the 3’ end of the coding region of exon 14. In some embodiments, the target sequence is located within about 1 to about 300 nucleotides, about 10 to about 250, about 20 to about 200, about 30 to about 150, about 40 to about 100, or about 50 nucleotides 5’ of the 3’ end of the coding region of exon 14. In some embodiments, the target sequence is at least partially within a targeted UTR sequence within the human DMPK gene. In some embodiments, the targeted UTR sequence is a portion within, contiguous with, or adjacent to a specified UTR of interest, which can be targeted by the compositions, systems, and methods described herein. In some embodiments, one or more UTR sequences are targeted. In some 91 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT embodiments, the target sequence is at least partially within the 3' UTR sequence within the human DMPK gene. In some embodiments, one or more of (CTG)
n repeats within a targeted UTR sequence are targeted. In some embodiments, the one or more of (CTG)
n repeats within a targeted UTR sequence are about (CTG)
50 to about (CTG)
5,000. [378] Nucleic acids, such as DNA and pre-mRNA, described herein can contain at least one intron and at least one exon, wherein as read in the 5’ to the 3’ direction of a nucleic acid strand, the 3’ end of an intron can be adjacent to the 5’ end of an exon, and wherein said intron and exon correspond for transcription purposes. If a nucleic acid strand contains more than one intron and exon, the 5’ end of the second intron is adjacent to the 3’ end of the first exon, and 5’ end of the second exon is adjacent to the 3’ end of the second intron. The junction between an intron and an exon can be referred to herein as a splice junction, wherein a 5’ splice site (SS) can refer to the +1/+2 position at the 5’ end of intron and a 3’SS can refer to the last two positions at the 3’ end of an intron. Alternatively, a 5’ SS can refer to the 5’ end of an exon and a 3’SS can refer to the 3’ end of an exon. In some embodiments, nucleic acids can contain one or more elements that act as a signal during transcription, splicing, and/or translation. In some embodiments, signaling elements include a 5’SS, a 3’SS, a premature stop codon, U1 and/or U2 binding sequences, and cis acting elements such as branch site (BS), polypyridine tract (PYT), exonic and intronic splicing enhancers (ESEs and ISEs) or silencers (ESSs and ISSs). In some embodiments, nucleic acids also comprise an untranslated region (UTR), such as a 5’ UTR or a 3’ UTR. In some embodiments, the start of an exon or intron is referred to interchangeably herein as the 5’ end of an exon or intron, respectively. Likewise, in some embodiments, the end of an exon or intron is referred to interchangeably herein as the 3’ end of an exon or intron, respectively. [379] In some embodiments, at least a portion of at least one target sequence is within 1, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 55 or more, about 60 or more, about 65 or more, about 70 or more, about 75 or more, about 80 or more, about 85 or more, about 90 or more, about 95 or more, about 100 or more, about 105 or more, about 110 or more, about 115 or more, about 120 or more, about 125 or more, about 130 or more, about 135 or more, about 140 or more, about 145 or more, or about 150 to about 300 nucleotides adjacent to: the 5’ end of an exon; the 3’ end of an exon; the 5’ end of an intron; the 3’ end of an intron; one or more signaling element comprising a 5’SS, a 3’SS, a premature stop codon, U1 binding sequence, U2 binding sequence, a BS, a PYT, ESE, an ISE, an ESS, an ISS; a 5’ UTR; a 3’ UTR; more than one of the foregoing, or a combination thereof. In some embodiments, the target nucleic acid comprises a target locus. In some embodiments, the target nucleic acid comprises more than one target loci. In some embodiments, the target nucleic acid comprises two target loci. Accordingly, in some embodiments, the target nucleic acid can comprise one or more target sequences. [380] In some embodiments, a target sequence that a guide nucleic acid binds is at least partially within a targeted UTR within the human DMPK gene, and wherein at least a portion of the target nucleic acid is within a sequence about 1 to about 300 nucleotides adjacent to: the start of a targeted UTR, the 92 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT end of a targeted UTR, or both. In some embodiments, at least a portion of the target sequence that a guide nucleic acid binds can comprise a sequence about 1 to about 300 nucleotides, about 10 to about 250, about 20 to about 200, about 30 to about 150, about 40 to about 100, or about 50 nucleotides adjacent to: the start of a targeted UTR, the end of a targeted UTR, or both. [381] In some embodiments, at least a portion of the target nucleic acid that a guide nucleic acid binds is within a sequence about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 55 or more, about 60 or more, about 65 or more, about 70 or more, about 75 or more, about 80 or more, about 85 or more, about 90 or more, about 95 or more, about 100 or more, about 105 or more, about 110 or more, about 115 or more, about 120 or more, about 125 or more, about 130 or more, about 135 or more, about 140 or more, about 145 or more, or about 150 or more nucleotides adjacent to: the start of a targeted UTR, the end of a targeted UTR, or both. [382] In some embodiments, compositions, systems, and methods described herein comprise an edited target nucleic acid which can describe a target nucleic acid wherein the target nucleic acid has undergone a change, for example, after contact with a polypeptide (e.g., an effector protein, an effector partner, a fusion protein, or a combination thereof). In some embodiments, the editing is an alteration in the sequence of the target nucleic acid. In some embodiments, the edited target nucleic acid comprises a nicked target strand or a nicked non-target strand. In some embodiments, the edited target nucleic acid comprises an insertion, deletion, or replacement of one or more nucleotides compared to the unedited target nucleic acid. In some embodiments, the editing is a mutation. In some embodiments, compositions, systems, and methods described herein comprise an edited target nucleic acid which can describe a target nucleic acid wherein the target nucleic acid (e.g., DMPK gene) has undergone a change (e.g., deletion of one or more (CTG)
n repeats within a UTR region of the DMPK gene) after contact with a polypeptide. Mutations [383] In some embodiments, target nucleic acids described herein comprise a mutation. In some embodiments, a composition, system or method described herein can be used to edit a target nucleic acid comprising a mutation such that the mutation is edited to be the wild-type nucleotide or nucleotide sequence. In some embodiments, a composition, system or method described herein can be used to detect a target nucleic acid comprising a mutation. In some embodiments, a mutation results in the insertion of at least one amino acid in a protein encoded by the target nucleic acid. In some embodiments, a mutation results in the deletion of at least one amino acid in a protein encoded by the target nucleic acid. In some embodiments, a mutation results in the substitution of at least one amino acid in a protein encoded by the target nucleic acid. A mutation that results in the deletion, insertion, or substitution of one or more amino acids of a protein encoded by the target nucleic acid results in misfolding of a protein encoded by the target nucleic acid. In some embodiments, a mutation results in a premature stop codon, thereby resulting in a truncation of the encoded protein. 93 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [384] Non-limiting examples of mutations are insertion-deletion (indel), a point mutation, single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation or variation, and frameshift mutations. In some embodiments, an indel mutation is an insertion or deletion of one or more nucleotides. In some embodiments, a point mutation comprises a substitution, insertion, or deletion. In some embodiments, a frameshift mutation occurs when the number of nucleotides in the insertion/deletion is not divisible by three, and it occurs in a protein coding region. In some embodiments, a chromosomal mutation can comprise an inversion, a deletion, a duplication, or a translocation of one or more nucleotides. In some embodiments, a copy number variation can comprise a gene amplification or an expanding trinucleotide repeat. In some embodiments, guide nucleic acids described herein hybridize to a region of the target nucleic acid comprising the mutation. The mutation may be located in a non-coding region or a coding region of a gene. The mutation may be located in a non-coding region or a coding region of a gene, wherein the gene is a target nucleic acid. A mutation may be in an open reading frame of a target nucleic acid. In some embodiments, guide nucleic acids described herein hybridize to a portion of the target nucleic acid comprising or adjacent to the mutation. In some embodiments, the mutation is associated with one or more of protein expression, protein activity, and protein structural stability. [385] In some embodiments, a target nucleic acid described herein comprises a mutation of one or more nucleotides. In some embodiments, the one or more nucleotides comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, the mutation comprises a deletion, insertion, and/or substitution of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 nucleotides. In some embodiments, the mutation comprises a deletion, insertion, and/or substitution of 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, 900 to 1000, 1 to 50, 1 to 100, 25 to 50, 25 to 100, 50 to 100, 100 to 500, 100 to 1000, or 500 to 1000 nucleotides. In some embodiments, the mutation is located in a non-coding region or a coding region of a gene, wherein the gene is a target nucleic acid. In some embodiments, a mutation is in an open reading frame of a target nucleic acid. In some embodiments, guide nucleic acids described herein hybridize to a portion of the target nucleic acid comprising or adjacent to the mutation. [386] In some embodiments, the target nucleic acid comprises one or more mutations. In some embodiments, the target nucleic acid comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more mutations as compared to the unmutated target nucleic acid. In some embodiments, the target nucleic acid comprises a sequence comprising one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more mutations as compared to the wildtype sequence. In some embodiments, the target nucleic acid comprises a mutation associated with a disease or disorder. 94 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [387] In some embodiments, target nucleic acids comprise a mutation, wherein the mutation is a SNP. In some embodiments, the single nucleotide mutation or SNP is associated with a phenotype of the sample or a phenotype of the organism from which the sample was taken. In some embodiments, the SNP is associated with altered phenotype from wild type phenotype. In some embodiments, a single nucleotide mutation, SNP, or deletion described herein is associated with a disease, such as a genetic disease. In some embodiments, the SNP is a synonymous substitution or a nonsynonymous substitution. In some embodiments, the nonsynonymous substitution is a missense substitution or a nonsense point mutation. In some embodiments, the synonymous substitution is a silent substitution. In some embodiments, the mutation is a deletion of one or more nucleotides. In some embodiments, the single nucleotide mutation, SNP, or deletion is associated with a disease such as a genetic disorder. In some embodiments, the mutation, such as a single nucleotide mutation, a SNP, or a deletion, is encoded in the sequence of a target nucleic acid from the germline of an organism or is encoded in a target nucleic acid from a diseased cell. [388] In some embodiments, the mutation is associated with a disease, such as a genetic disorder. In some embodiments, the mutation is encoded in the sequence of a target nucleic acid from the germline of an organism or is encoded in a target nucleic acid from a diseased cell. In some embodiments, a target nucleic acid described herein comprises a mutation associated with a disease. In some examples, a mutation associated with a disease refers to a mutation whose presence in a subject indicates that the subject is susceptible to or suffers from, a disease, disorder, condition, or syndrome. In some examples, a mutation associated with a disease refers to a mutation which causes, contributes to the development of, or indicates the existence of the disease, disorder, condition, or syndrome. In some embodiments, a mutation associated with a disease is also refer to any mutation which generates transcription or translation products at an abnormal level, or in an abnormal form, in cells affected by a disease relative to a control without the disease. In some examples, a mutation associated with a disease refers to a mutation whose presence in a subject indicates that the subject is susceptible to, or suffers from, a disease, disorder, or pathological state. In some embodiments, a mutation associated with a disease, comprises the co-occurrence of a mutation and the phenotype of a disease. In some embodiments, the mutation occurs in a gene, wherein transcription or translation products from the gene occur at a significantly abnormal level or in an abnormal form in a cell or subject harboring the mutation as compared to a non-disease control subject not having the mutation. In some embodiments, a target nucleic acid described herein comprises a mutation associated with a disease, wherein the target nucleic acid is any one of the target nucleic acids recited in TABLE 9 or 9.1. In some embodiments, the disease, disorder, condition, syndrome or pathological state comprises any one of the diseases or syndromes recited in TABLE 10. [389] The mutation may cause a disease. The disease can comprise, at least in part, an inherited disorder, a neurological disorder, or both. The disease can comprise, at least in part, an inherited disorder. The disease can comprise, at least in part, a neurological disorder. In some embodiments, the neurological disorder is a neuromuscular disorder. In some embodiments, the neuromuscular disorder comprises: muscular dystrophy (MD); myotonic dystrophy (DM); myotonic dystrophy type 1 (DM1); dystrophia myotonica; myotonia 95 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT atrophica; myotonia dystrophica; Steinert disease; Curschmann–Batten–Steinert syndrome; hypotonia; cardiomyopathy; or MD-associated cardiomyopathy. [390] In some embodiments, a composition, system or method described herein can be used to edit a target nucleic acid (e.g., DMPK gene) comprising a mutation such that the mutation is edited to be the wild- type nucleotide or nucleotide sequence. Detection of Target Nucleic Acids [391] Described herein are systems and methods for detecting the presence of a target nucleic acid in a sample. In some embodiments, a target nucleic acid described herein comprises a mutation associated with a disease, wherein the target nucleic acid is any one of the target nucleic acids recited in TABLE 9 or 9.1. In some embodiments, a target nucleic acid described herein comprises a mutation associated with a disease, wherein the disease is any one of the diseases recited in TABLE 10. In some embodiments, a target nucleic acid is in a cell. In some embodiments, the cell is a eukaryotic cell, a mammalian cell, a human cell. In some embodiments, the human cell is a: muscle cell, cardiac cell, visceral cell, cardiac muscle cell, smooth muscle cell, cardiomyocyte, nodal cardiac muscle cell, smooth muscle cell, visceral muscle cell, skeletal muscle cell, myocyte, red (or slow) skeletal muscle cell, white (fast) skeletal muscle cell, intermediate skeletal muscle, muscle satellite cell, muscle stem cell, myoblast, muscle progenitor cell, induced pluripotent stem cell (iPS), or a cell derived from an iPS cell, modified to have its gene edited and differentiated into myoblasts, muscle progenitor cells, muscle satellite cells, muscle stem cells, skeletal muscle cells, cardiac muscle cells or smooth muscle cells. In some embodiments, the muscle can be skeletal muscle. In certain instances, skeletal muscles include the following: abductor digiti minimi (foot), abductor digiti minimi (hand), abductor hallucis, abductor pollicis brevis, abductor pollicis longus, adductor brevis, adductor hallucis, adductor longus, adductor magnus, adductor pollicis, anconeus, articularis cubiti, articularis genu, aryepiglotticus, auricularis, biceps brachii, biceps femoris, brachialis, brachioradialis, buccinator, bulbospongiosus, constrictor of pharynx -inferior, constrictor of pharynx -middle, constrictor of pharynx -superior, coracobrachialis, corrugator supercilii, cremaster, cricothyroid, dartos, deep transverse perinei, deltoid, depressor anguli oris, depressor labii inferioris, diaphragm, digastric, digastric (anterior view), erector spinae - spinalis, erector spinae - iliocostalis, erector spinae - longissimus, extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi ulnaris, extensor digiti minimi (hand), extensor digitorum (hand), extensor digitorum brevis (foot), extensor digitorum longus (foot), extensor hallucis brevis, extensor hallucis longus, extensor indicis, extensor pollicis brevis, extensor pollicis longus, external oblique abdominis, flexor carpi radialis, flexor carpi ulnaris, flexor digiti minimi brevis (foot), flexor digiti minimi brevis (hand), flexor digitorum brevis, flexor digitorum longus (foot), flexor digitorum profundus, flexor digitorum superficialis, flexor hallucis brevis, flexor hallucis longus, flexor pollicis brevis, flexor pollicis longus, frontalis, gastrocnemius, gemellus inferior, gemellus superior, genioglossus, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus, gracilis, hyoglossus, iliacus, inferior oblique, inferior rectus, infraspinatus, intercostals external, intercostals innermost, intercostals internal, internal oblique abdominis, interossei - dorsal of hand, interossei -dorsal of foot, interossei- palmar of hand, 96 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT interossei - plantar of foot, interspinales, intertransversarii, intrinsic muscles of tongue, ishiocavernosus, lateral cricoarytenoid, lateral pterygoid, lateral rectus, latissimus dorsi, levator anguli oris, levator ani- coccygeus, levator ani - iliococcygeus, levator ani-pubococcygeus, levator ani-puborectalis, levator ani- pubovaginalis, levator labii superioris, levator labii superioris, alaeque nasi, levator palpebrae superioris, levator scapulae, levator veli palatini, levatores costarum, longus capitis, longus colli, lumbricals of foot, lumbricals of hand, masseter, medial pterygoid, medial rectus, mentalis, m. uvulae, mylohyoid, nasalis, oblique arytenoid, obliquus capitis inferior, obliquus capitis superior, obturator externus, obturator internus (A), obturator internus (B), omohyoid, opponens digiti minimi (hand), opponens pollicis, orbicularis oculi, orbicularis oris, palatoglossus, palatopharyngeus, palmaris brevis, palmaris longus, pectineus, pectoralis major, pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius, piriformis (A), piriformis (B), plantaris, platysma, popliteus, posterior cricoarytenoid, procerus, pronator quadratus, pronator teres, psoas major, psoas minor, pyramidalis, quadratus femoris, quadratus lumborum, quadratus plantae, rectus abdominis, rectus capitus anterior, rectus capitus lateralis, rectus capitus posterior major, rectus capitus posterior minor, rectus femoris, rhomboid major, rhomboid minor, risorius, salpingopharyngeus, sartorius, scalenus anterior, scalenus medius, scalenus minimus, scalenus posterior, semimembranosus, semitendinosus, serratus anterior, serratus posterior inferior, serratus posterior superior, soleus, sphincter ani, sphincter urethrae, splenius capitis, splenius cervicis, stapedius, sternocleidomastoid, sternohyoid, sternothyroid, styloglossus, stylohyoid, stylohyoid (anterior view), stylopharyngeus, subclavius, subcostalis, subscapularis, superficial transverse perinei, superior oblique, superior rectus, supinator, supraspinatus, temporalis, temporoparietalis, tensor fasciae lata, tensor tympani, tensor veli palatini, teres major, teres minor, thyro-arytenoid & vocalis, thyro-epiglotticus, thyrohyoid, tibialis anterior, tibialis posterior, transverse arytenoid, transversospinalis -multifidus, transversospinalis -rotatores, transversospinalis -semispinalis, transversus abdominis, transversus thoracis, trapezius, triceps, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major, or zygomaticus minor. In some instances, the cell is a myocyte. In some instances, the cell is a muscle cell. In some instances, the muscle cell is a skeletal muscle cell. In some instances, the skeletal muscle cell is a red (slow) skeletal muscle cell, a white (fast) skeletal muscle cell or an intermediate skeletal muscle cell. [392] In some embodiments, an effector protein-guide nucleic acid complex comprises high selectivity for a target sequence. In some embodiments, an RNP comprise a selectivity of at least 200:1, 100:1, 50:1, 20:1, 10:1, or 5:1 for a target nucleic acid over a single nucleotide variant of the target nucleic acid. In some embodiments, an RNP comprises a selectivity of at least 5:1 for a target nucleic acid over a single nucleotide variant of the target nucleic acid. [393] By leveraging such effector protein selectivity, some methods described herein detects a target nucleic acid present in the sample in various concentrations or amounts as a target nucleic acid population. In some embodiments, the method detects at least 2 target nucleic acid populations. In some embodiments, the method detects at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 target nucleic acid populations. In some embodiments, the method detects 3 to 50, 5 to 40, or 10 to 25 target nucleic acid populations. In some 97 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT embodiments, the method detects at least 2 individual target nucleic acids. In some embodiments, the method detects at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 individual target nucleic acids. In some embodiments, the method detects 1 to 10,000, 100 to 8000, 400 to 6000, 500 to 5000, 1000 to 4000, or 2000 to 3000 individual target nucleic acids. In some embodiments, the method detects target nucleic acid present at least at one copy per 10 non-target nucleic acids, 10
2 non-target nucleic acids, 10
3 non-target nucleic acids, 10
4 non-target nucleic acids, 10
5 non-target nucleic acids, 10
6 non-target nucleic acids, 10
7 non-target nucleic acids, 10
8 non-target nucleic acids, 10
9 non-target nucleic acids, or 10
10 non-target nucleic acids. [394] In some embodiments, a target nucleic acid is an amplified nucleic acid of interest. In some embodiments, the nucleic acid of interest is any nucleic acid disclosed herein or from any sample as disclosed herein. In some embodiments, the nucleic acid of interest is DNA. In some embodiments, the nucleic acid of interest is an RNA. In some embodiments, the nucleic acid of interest is an RNA that is reverse transcribed before amplification. In some embodiments, the target nucleic acid is an amplicon of a target nucleic acid (DNA or RNA) generated via amplification (with or without reverse transcription). In some embodiments, the target nucleic acid is an amplicon of a target nucleic acid (DNA or RNA) generated via amplification that is reverse transcribed before amplification. [395] In some embodiments, target nucleic acids activate an effector protein to initiate sequence- independent cleavage of a nucleic acid-based reporter (e.g., a reporter comprising a DNA sequence, or a reporter comprising DNA and/or RNA). For example, an effector protein of the present disclosure is activated by a target nucleic acid to cleave reporters having a DNA (also referred to herein as a “DNA reporter”). Alternatively, an effector protein of the present disclosure is activated by a target nucleic acid to cleave reporters having an RNA (also referred to herein as a “RNA reporter”). In some embodiments, the nucleic acid-based reporter comprises a single-stranded DNA labelled with a detection moiety or any DNA reporter as disclosed herein. [396] Further description of editing or detecting a target nucleic acid in a gene of interest can be found in more detail in Kim et al., “Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA- RNA guide”, Nucleic Acids Res. 2020 Sep 4;48(15):8601-8616; Wang et al., “Specificity profiling of CRISPR system reveals greatly enhanced off-target gene editing”, Scientific Reports volume 10, Article number: 2269 (2020); Tuladhar et al., “CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation”, Nature Communications volume 10, Article number: 4056 (2019); Dong et al., “Genome-Wide Off-Target Analysis in CRISPR-Cas9 Modified Mice and Their Offspring”, G3, Volume 9, Issue 11, 1 November 2019, Pages 3645–3651; Winter et al., “Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus α-hemolysin-mediated toxicity”, Scientific Reports volume 6, Article number: 24242 (2016); and Ma et al., “A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death”, Cell Rep.2015 Jul 28;12(4):673-83, which are hereby incorporated by reference in their entirety. Samples 98 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [397] Various sample types comprising a target nucleic acid of interest are consistent with the present disclosure. In some embodiments, the samples comprise a target nucleic acid for detection. In some embodiments, the detection of the target nucleic indicates an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry and are compatible with the reagents and support mediums as described herein. Generally, a sample from an individual or an animal or an environmental sample is obtained for testing presence of a disease, cancer, genetic disorder, or any mutation of interest. [398] In some embodiments, a sample comprises a target nucleic acid from 0.05% to 20% of total nucleic acids in the sample. In some embodiments, the target nucleic acid is 0.1% to 10% of the total nucleic acids in the sample. In some embodiments, the target nucleic acid is 0.1% to 5% of the total nucleic acids in the sample. In some embodiments, the target nucleic acid is 0.1% to 1% of the total nucleic acids in the sample. In some embodiments, the target nucleic acid is in any amount less than 100% of the total nucleic acids in the sample. In some embodiments, the target nucleic acid is 100% of the total nucleic acids in the sample. In some embodiments, the sample comprises a portion of the target nucleic acid and at least one nucleic acid comprising less than 100% sequence identity to the portion of the target nucleic acid but no less than 50% sequence identity to the portion of the target nucleic acid. For example, the portion of the target nucleic acid comprises a mutation as compared to at least one nucleic acid comprising less than 100% sequence identity to the portion of the target nucleic acid but no less than 50% sequence identity to the portion of the target nucleic acid. In some embodiments, the portion of the target nucleic acid comprises a single nucleotide mutation as compared to at least one nucleic acid comprising less than 100% sequence identity to the portion of the target nucleic acid but no less than 50% sequence identity to the portion of the target nucleic acid. [399] In some embodiments, a sample comprises target nucleic acid populations at different concentrations or amounts. In some embodiments, the sample has at least 2 target nucleic acid populations. In some embodiments, the sample has at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 target nucleic acid populations. In some embodiments, the sample has 3 to 50, 5 to 40, or 10 to 25 target nucleic acid populations. [400] In some embodiments, a sample has at least 2 individual target nucleic acids. In some embodiments, the sample has at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 individual target nucleic acids. In some embodiments, the sample comprises 1 to 10,000, 100 to 8000, 400 to 6000, 500 to 5000, 1000 to 4000, or 2000 to 3000 individual target nucleic acids. [401] In some embodiments, a sample comprises one copy of target nucleic acid per 10 non-target nucleic acids, 10
2 non-target nucleic acids, 10
3 non-target nucleic acids, 10
4 non-target nucleic acids, 10
5 non-target nucleic acids, 10
6 non-target nucleic acids, 10
7 non-target nucleic acids, 10
8 non-target nucleic acids, 10
9 non-target nucleic acids, or 10
10 non-target nucleic acids. 99 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [402] In some embodiments, samples comprise a target nucleic acid at a concentration of less than 1 nM, less than 2 nM, less than 3 nM, less than 4 nM, less than 5 nM, less than 6 nM, less than 7 nM, less than 8 nM, less than 9 nM, less than 10 nM, less than 20 nM, less than 30 nM, less than 40 nM, less than 50 nM, less than 60 nM, less than 70 nM, less than 80 nM, less than 90 nM, less than 100 nM, less than 200 nM, less than 300 nM, less than 400 nM, less than 500 nM, less than 600 nM, less than 700 nM, less than 800 nM, less than 900 nM, less than 1 µM, less than 2 µM, less than 3 µM, less than 4 µM, less than 5 µM, less than 6 µM, less than 7 µM, less than 8 µM, less than 9 µM, less than 10 µM, less than 100 µM, or less than 1 mM. In some embodiments, the sample comprises a target nucleic acid at a concentration of 1 nM to 2 nM, 2 nM to 3 nM, 3 nM to 4 nM, 4 nM to 5 nM, 5 nM to 6 nM, 6 nM to 7 nM, 7 nM to 8 nM, 8 nM to 9 nM, 9 nM to 10 nM, 10 nM to 20 nM, 20 nM to 30 nM, 30 nM to 40 nM, 40 nM to 50 nM, 50 nM to 60 nM, 60 nM to 70 nM, 70 nM to 80 nM, 80 nM to 90 nM, 90 nM to 100 nM, 100 nM to 200 nM, 200 nM to 300 nM, 300 nM to 400 nM, 400 nM to 500 nM, 500 nM to 600 nM, 600 nM to 700 nM, 700 nM to 800 nM, 800 nM to 900 nM, 900 nM to 1 µM, 1 µM to 2 µM, 2 µM to 3 µM, 3 µM to 4 µM, 4 µM to 5 µM, 5 µM to 6 µM, 6 µM to 7 µM, 7 µM to 8 µM, 8 µM to 9 µM, 9 µM to 10 µM, 10 µM to 100 µM, 100 µM to 1 mM, 1 nM to 10 nM, 1 nM to 100 nM, 1 nM to 1 µM, 1 nM to 10 µM, 1 nM to 100 µM, 1 nM to 1 mM, 10 nM to 100 nM, 10 nM to 1 µM, 10 nM to 10 µM, 10 nM to 100 µM, 10 nM to 1 mM, 100 nM to 1 µM, 100 nM to 10 µM, 100 nM to 100 µM, 100 nM to 1 mM, 1 µM to 10 µM, 1 µM to 100 µM, 1 µM to 1 mM, 10 µM to 100 µM, 10 µM to 1 mM, or 100 µM to 1 mM. In some embodiments, the sample comprises a target nucleic acid at a concentration of 20 nM to 200 µM, 50 nM to 100 µM, 200 nM to 50 µM, 500 nM to 20 µM, or 2 µM to 10 µM. In some embodiments, the target nucleic acid is not present in the sample. [403] In some embodiments, samples comprise fewer than 10 copies, fewer than 100 copies, fewer than 1,000 copies, fewer than 10,000 copies, fewer than 100,000 copies, or fewer than 1,000,000 copies of a target nucleic acid. In some embodiments, the sample comprises 10 copies to 100 copies, 100 copies to 1,000 copies, 1,000 copies to 10,000 copies, 10,000 copies to 100,000 copies, 100,000 copies to 1,000,000 copies, 10 copies to 1,000 copies, 10 copies to 10,000 copies, 10 copies to 100,000 copies, 10 copies to 1,000,000 copies, 100 copies to 10,000 copies, 100 copies to 100,000 copies, 100 copies to 1,000,000 copies, 1,000 copies to 100,000 copies, or 1,000 copies to 1,000,000 copies of a target nucleic acid. In some embodiments, the sample comprises 10 copies to 500,000 copies, 200 copies to 200,000 copies, 500 copies to 100,000 copies, 1,000 copies to 50,000 copies, 2,000 copies to 20,000 copies, 3,000 copies to 10,000 copies, or 4,000 copies to 8,000 copies. In some embodiments, the target nucleic acid is not present in the sample. [404] In some embodiments, the sample is a biological sample, an environmental sample, or a combination thereof. Non-limiting examples of biological samples are blood, serum, plasma, saliva, urine, mucosal sample, peritoneal sample, cerebrospinal fluid, gastric secretions, nasal secretions, sputum, pharyngeal exudates, urethral or vaginal secretions, an exudate, an effusion, and a tissue sample (e.g., a biopsy sample). In some embodiments, a tissue sample from a subject is dissociated or liquified prior to application to detection system of the present disclosure. Non-limiting examples of environmental samples 100 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT are soil, air, or water. In some embodiments, an environmental sample is taken as a swab from a surface of interest or taken directly from the surface of interest. [405] In some embodiments, the sample is a raw (unprocessed, unedited, unmodified) sample. In some embodiments, raw samples are applied to a system for detecting or editing a target nucleic acid, such as those described herein. In some embodiments, the sample is diluted with a buffer or a fluid or concentrated prior to its application to the system or be applied neat to the detection system. Sometimes, the sample contains no more 20 µl of buffer or fluid. The sample, in some embodiments, is contained in no more than 0.01, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 3540, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500 µl, or any of value 0.01 µl to 500 µl, 0.1 µL to 100 µL, or more preferably 1 µL to 50 µL of buffer or fluid. Sometimes, the sample is contained in more than 500 µl. In some embodiments, the compositions, systems, and methods disclosed herein are compatible with the buffers or fluid disclosed herein. [406] In some embodiments, the sample is taken from a human. The sample can comprise one or more cells. The sample can be a tissue sample (e.g., biopsy sample). In some embodiments, the cell is a muscle cell. The sample comprises nucleic acids from a cell lysate from a muscle cell. The sample comprises nucleic acids from a cell lysate from a neuron, brain stem neuron, cerebrum neuron, cardiac muscle cell, smooth or visceral muscle cell, or a skeletal muscle cell. In some embodiments, the sample comprises nucleic acids expressed from a cell. [407] In some embodiments, samples are used for diagnosing a disease. In some embodiments the disease is a genetic disorder. In some embodiments, the sample used for genetic testing comprises at least one target nucleic acid that hybridizes to a guide nucleic acid of the reagents described herein. The target nucleic acid, in some embodiments, comprises a portion of a gene comprising a mutation associated with a genetic disease or a gene whose expression is associated with a genetic disease. Sometimes, the target nucleic acid encodes a disease biomarker, such as a gene mutation. In some embodiments, the target nucleic acid is a portion of a nucleic acid from a genomic locus, any DNA amplicon of, a reverse transcribed mRNA, or a cDNA from a locus of at least one of the genes recited in TABLE 9. Any region of the aforementioned gene loci is probed for a mutation or deletion using the compositions and methods disclosed herein. For example, in the DMPK gene locus, the compositions and methods for detection disclosed herein can be used to detect a single nucleotide polymorphism or a deletion. In some embodiments, the gene is DMPK. In some embodiments, the contacting occurs in vitro. In some embodiments, the contacting occurs in vivo. In some embodiments, the contacting occurs ex vivo. In some embodiments, the target nucleic acid comprises a portion of a nucleic acid from a genomic locus, any DNA amplicon of, a reverse transcribed mRNA, or a cDNA from a locus of DMPK. [408] In some embodiments, the genetic disorder is myotonic dystrophy (DM), myotonic dystrophy type 1 (DM1), Steinert disease, Curschmann–Batten–Steinert syndrome, neural shrinkage, myelin degeneration, axonal neuropathy, or MD-associated cardiomyopathy. The target nucleic acid, in some embodiments, is from a gene with a mutation associated with a genetic disorder, from a gene whose overexpression is associated with a genetic disorder, from a gene associated with abnormal cellular growth resulting in a 101 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT genetic disorder, or from a gene associated with abnormal cellular metabolism resulting in a genetic disorder. In some embodiments, the target nucleic acid is a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed mRNA, a DNA amplicon of or a cDNA from a locus of at least one of a gene recited in TABLE 9. In some embodiments, the target nucleic acid is encoded by a gene described in TABLE 9. In some embodiments, the target nucleic acid is encoded by a gene described in TABLE 9 comprising a mutation. In some embodiments, the target nucleic acid is any of the target nucleic acids described in TABLE 9.1. In some embodiments, the target nucleic acid is in any one of the locations described in TABLE 9.2. [409] In some embodiments, a sample used for phenotyping testing comprise at least one target nucleic acid that hybridizes to a guide nucleic acid of the reagents described herein. The target nucleic acid, in some embodiments, is a nucleic acid encoding a sequence associated with a phenotypic trait. In some embodiments, a sample used for genotyping testing comprises at least one target nucleic acid that hybridizes to a guide nucleic acid of the reagents described herein. A target nucleic acid, in some embodiments, is a nucleic acid encoding a sequence associated with a genotype of interest. In some embodiments, a sample used for ancestral testing comprise at least one target nucleic acid that hybridizes to a guide nucleic acid of the reagents described herein. A target nucleic acid, in some embodiments, is a nucleic acid encoding a sequence associated with a geographic region of origin or ethnic group. In some embodiments, a sample is used for identifying a disease status. For example, a sample is any sample described herein, and is obtained from a subject for use in identifying a disease status of a subject. In some embodiments, the disease is a genetic disorder. In some embodiments, a method comprises obtaining a serum sample from a subject; and identifying a disease status of the subject. Compositions [410] Disclosed herein are compositions comprising one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or a combination thereof) described herein or nucleic acids encoding the one or more polypeptides, one or more guide nucleic acids described herein or nucleic acids encoding the one or more guide nucleic acids described herein, or a combination thereof. In some embodiments, repeat sequences of the one or more guide nucleic acids are capable of interacting with the one or more of the effector proteins. In some embodiments, spacer sequences of the one or more guide nucleic acids hybridizes with a target sequence of a target nucleic acid. In some embodiments, the compositions comprise one or more donor nucleic acids described herein. In some embodiments, the compositions are capable of editing a target nucleic acid in a cell or a subject. In some embodiments, the compositions are capable of editing a target nucleic acid or the expression thereof in a cell, in a tissue, in an organ, in vitro, in vivo, or ex vivo. In some embodiments, the compositions are capable of editing a target nucleic acid in a sample comprising the target nucleic. [411] In some embodiments, compositions described herein comprise components for modifying or editing at least one target nucleic acid associated with a disease described herein or the expression thereof. 102 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT In some embodiments, the target nucleic acid is any one of the target nucleic acids listed in TABLE 9 or 9.1. In some embodiments, the target nucleic acid is a human DMPK gene. In some embodiments, the target nucleic acid comprises a mutation relative to the wild-type DMPK gene. In some embodiments, the compositions modify or edit one or more nucleotides of a target nucleic acid. In some embodiments, modifying comprises cleaving at least one strand of a target nucleic acid, deleting or excising one or more nucleotides of the target nucleic acid, inserting one or more nucleotides into the target nucleic acid, substituting one or more nucleotides of the target nucleic acid with one or more alternative nucleotides, or combinations thereof. In some embodiments, modifying comprises deleting or excising one or more nucleotides of a target nucleic acid. In some embodiments, the compositions comprise an additional engineered guide nucleic acid, or a nucleic acid encoding an additional engineered guide nucleic acid at least a portion of which hybridizes to a different target sequence of the target nucleic acid than the engineered guide nucleic acid. In some embodiments, the compositions cleave two loci of a target nucleic acid, and wherein the composition excises one or more nucleotides between the two loci of the target nucleic acid. In some embodiments, modifying comprises deleting or excising one or more nucleotides of a target nucleic acid, wherein the one or more nucleotides are located in an untranslated region, protein coding region, an exon, an intron, a gene regulatory region, coding sequences thereof, or combinations thereof. In some embodiments, the one or more nucleotides are located in any one of the locations listed in TABLE 9.2. In some embodiments, the one or more nucleotides are located in an untranslated region (UTR) of a target nucleic acid. In some embodiments, the UTR is the 3’ UTR of a target nucleic acid (e.g., DMPK gene). In some embodiments, the 3’ UTR of a target nucleic acid is downstream of the coding region of exon 14 of the DMPK gene. In some embodiments, the 3’ UTR of a target nucleic acid comprises expansion of a (CTG)
n repeat. [412] As described herein, in some embodiments, the target nucleic acid comprises a mutation relative to the wild-type DMPK gene, wherein the mutation comprises an expansion of a (CTG)
n repeat. In some embodiments, the expansion of a (CTG)
n repeat is in the 3’ UTR of a target nucleic acid. In some embodiments, the expansion of the (CTG)
n are greater than about (CTG)
30, (CTG)
40, or about (CTG)
50. In some embodiments, the expansion of the (CTG)
n are about (CTG)
50 to about (CTG)
5,000. In some embodiments, the mutation is associated with a disease, wherein the disease is any one of the diseases listed in TABLE 10. [413] In some embodiments, compositions described herein comprise one or more effector proteins described herein and one or more guide nucleic acids described herein, wherein the one or more effector proteins edit (e.g., cleave) or modify the target nucleic acid wherein the target nucleic acid comprises the (CTG)
n repeat region of the DMPK gene. In some embodiments, effector protein cleaves the target nucleic acid, reducing or removing the (CTG)
n repeat mutation. In some embodiments, effector protein cleaves the target nucleic acid, reducing or removing the (CTG)
n repeat region, thus reducing the transcription of DMPK RNA and/or the overall amount of RNA transcript. 103 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [414] In some embodiments, compositions described herein comprise a polypeptide (e.g., effector protein) and guide nucleic acid, wherein the polypeptide is a variant of SEQ ID NO: 1, and wherein the guide nucleic acid comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 43-49 or 50-65. In some embodiments, the guide nucleic acid comprises a repeat sequence, wherein the repeat sequence is a nucleotide sequence that is at least 85% identical to SEQ ID NO: 17. In some embodiments, the guide nucleic acid comprises a spacer sequence, wherein a spacer sequence is a nucleotide sequence that is at least 85% identical to SEQ ID NO: 19-25. [415] In some embodiments, compositions described herein comprise a polypeptide (e.g., effector protein) and guide nucleic acid, wherein the polypeptide is a variant of SEQ ID NO: 2, and wherein the guide nucleic acid comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 50-65. In some embodiments, the guide nucleic acid comprises a repeat sequence, wherein the repeat sequence is a nucleotide sequence that is at least 85% identical to SEQ ID NO: 18. In some embodiments, the guide nucleic acid comprises a spacer sequence, wherein a spacer sequence is a nucleotide sequence that is at least 85% identical to SEQ ID NO: 26-41. [416] In some embodiments, compositions described herein comprise systems for editing, modifying or detecting at least one target nucleic acid as described herein. [417] In some embodiments, compositions described herein comprise plasmids described herein, viral vectors described herein, non-viral vectors described herein, or combinations thereof. In some embodiments, compositions described herein comprise the viral vectors. In some embodiments, compositions described herein comprise an AAV. In some embodiments, compositions described herein comprise liposomes (e.g., cationic lipids or neutral lipids), dendrimers, lipid nanoparticle (LNP), or cell- penetrating peptides. In some embodiments, compositions described herein comprise an LNP. Pharmaceutical Compositions [418] Described herein are formulations of introducing compositions or components of a system described herein to a host. [419] In some embodiments, compositions described herein are pharmaceutical compositions. In some embodiments, the pharmaceutical compositions comprise compositions described herein or systems described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable salt, one or more of a vehicle, adjuvant, excipient, or carrier, such as a filler, disintegrant, a surfactant, a binder, a lubricant, or combinations thereof. Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy, Lippincott Williams & Wilkins, Philadelphia; Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York; and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick, 2015, CRC Press, Boca Raton disclose various carriers used in formulating pharmaceutically acceptably compositions and known techniques for the preparation thereof. Non-limiting examples of pharmaceutically acceptable carriers and diluents suitable for the pharmaceutical compositions disclosed herein include buffers (e.g., neutral buffered saline, 104 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT phosphate buffered saline); carbohydrates (e.g., glucose, mannose, sucrose, dextran, mannitol); polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA, glutathione); adjuvants (e.g., aluminum hydroxide); surfactants (Polysorbate 80, Polysorbate 20, or Pluronic F68); glycerol; sorbitol; mannitol; polyethyleneglycol; and preservatives. In some embodiments, the vector is formulated for delivery through injection by a needle carrying syringe. In some embodiments, the composition is formulated for delivery by electroporation. In some embodiments, the composition is formulated for delivery by chemical method. In some embodiments, the pharmaceutical compositions comprise a virus vector or a non-viral vector. [420] Pharmaceutical compositions described herein comprise a salt. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is a potassium salt. In some embodiments, the salt is a magnesium salt. In some embodiments, the salt is NaCl. In some embodiments, the salt is KNO
3. In some embodiments, the salt is Mg
2+ SO
4 2−. [421] Pharmaceutical compositions described herein are in the form of a solution (e.g., a liquid). In some embodiments, the solution is formulated for injection, e.g., intravenous or subcutaneous injection. In some embodiments, the pH of the solution is about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9. In some embodiments, the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5. In some cases, the pH of the solution is less than 7. In some cases, the pH is greater than 7. Systems [422] Disclosed herein, in some aspects, are systems for editing, modifying, or detecting at least one target nucleic acid, comprising any one of the effector proteins described herein. In some embodiments, systems comprise a guide nucleic acid described herein. In some embodiments, systems comprise a guide nucleic acid and an additional nucleic acid. In some embodiments, systems comprise one or more components having a guide nucleic acid. In some embodiments, systems comprise one or more components having a guide nucleic acid and an additional nucleic acid. In some embodiments, systems are used for detecting a target nucleic acid. In some embodiments, systems are used for modifying or editing a target nucleic acid. In some embodiments, systems comprise an effector protein described herein, one or more guide nucleic acids, an additional nucleic acid, a reagent, a support medium, or combinations thereof. In some embodiments, systems comprise compositions, a solution, a buffer, a reagent, a support medium, or combinations thereof. In some embodiments, systems further comprise a donor nucleic acid as disclosed herein. In some embodiments, systems or system components described herein are comprised in a single composition. [423] In some embodiments, systems comprise a fusion protein described herein. In some embodiments, effector proteins comprise an amino acid sequence that is a variant of any one of the amino acid sequences 105 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT selected from TABLE 1. In some embodiments, the amino acid sequence of the effector protein is a variant of any one of the amino acid sequences selected from TABLE 1. [424] In some embodiments, solutions, compositions, systems, and methods comprise 0.01 µL, 0.02 µL, 0.03 µL, 0.04 µL, 0.05 µL, 0.06 µL, 0.07 µL, 0.08 µL, 0.09 µL, 0.1 µL, 0.2 µL, 0.3 µL, 0.4 µL, 0.5 µL, 0.6 µL, 0.7 µL, 0.8 µL, 0.9 µL, 1 µL, 2 µL, 3 µL, 4 µL, 5 µL, 6 µL, 7 µL, 8 µL, 9 µL, 10 µL, 20 µL, 30 µL, 40 µL, 50 µL, 60 µL, 70 µL, 80 µL, 90 µL, 100 µL, 150 µL, 200 µL, 250 µL, 300 µL, 350 µL, 400 µL, 450 µL, 500 µL, or more, effector proteins, or nucleic acids encoding the effector proteins, as described herein. In some embodiments, solutions, compositions, systems, and methods comprise 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, or more, effector proteins, or nucleic acids encoding the effector proteins, as described herein. In some embodiments, solutions, compositions, systems, and methods, comprise 1 µM, 2 µM, 3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM, 9 µM, 10 µM, 20 µM, 30 µM, 40 µM, 50 µM, 60 µM, 70 µM, 80 µM, 90 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 350 µM, 400 µM, 450 µM, 500 µM, or more, effector proteins, or nucleic acids encoding the effector proteins, as described herein. In some embodiments, solutions, compositions, systems, and methods comprise 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, or more, effector proteins, or nucleic acids encoding the effector proteins, as described herein. [425] In some embodiments, solutions, compositions, systems, and methods comprise 0.01 µL, 0.02 µL, 0.03 µL, 0.04 µL, 0.05 µL, 0.06 µL, 0.07 µL, 0.08 µL, 0.09 µL, 0.1 µL, 0.2 µL, 0.3 µL, 0.4 µL, 0.5 µL, 0.6 µL, 0.7 µL, 0.8 µL, 0.9 µL, 1 µL, 2 µL, 3 µL, 4 µL, 5 µL, 6 µL, 7 µL, 8 µL, 9 µL, 10 µL, 20 µL, 30 µL, 40 µL, 50 µL, 60 µL, 70 µL, 80 µL, 90 µL, 100 µL, 150 µL, 200 µL, 250 µL, 300 µL, 350 µL, 400 µL, 450 µL, 500 µL, or more, guide nucleic acids, or nucleic acids encoding the guide nucleic acids, as described herein. In some embodiments, solutions, compositions, systems, and methods comprise 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, or more, guide nucleic acids, or nucleic acids encoding the guide nucleic acids, as described herein. In some embodiments, solutions, compositions, systems, and methods comprise 1 µM, 2 µM, 3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM, 9 µM, 10 µM, 20 µM, 30 µM, 40 µM, 50 µM, 60 µM, 70 µM, 80 µM, 90 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 350 µM, 400 µM, 450 µM, 500 µM, or more, guide nucleic acids, or nucleic acids encoding the guide nucleic acids, as described herein. In some embodiments, solutions, compositions, systems, and methods comprise 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, or more, guide nucleic acids, or nucleic acids encoding the guide nucleic acids, as described herein. 106 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [426] In some embodiments, systems are used for detecting the presence of a target nucleic acid associated with or causative of a disease, such as cancer, a genetic disorder, or an infection. In some embodiments, systems are useful for phenotyping, genotyping, or determining ancestry. Unless specified otherwise, systems comprise kits. In some embodiments, the systems comprising kits are referred to as kits. Unless specified otherwise, systems comprise devices. In some embodiments, the systems comprising the devices are referred to as devices. In some embodiments, systems described herein are provided in the form of a companion diagnostic assay or device, a point-of-care assay or device, or an over-the-counter diagnostic assay/device. Unless specified otherwise, systems described herein are used in methods for detecting the presence of a target nucleic acid. [427] In some embodiments, reagents and effector proteins of various systems are provided in a reagent chamber or on a support medium. Alternatively, the reagent and/or effector protein, in some embodiments, are contacted with the reagent chamber or the support medium by the individual using the system. An exemplary reagent chamber is a test well or container. In some embodiments, the opening of the reagent chamber is large enough to accommodate the support medium. Optionally, the system comprises a buffer and a dropper. In some embodiments, the buffer is provided in a dropper bottle for ease of dispensing. In some embodiments, the dropper is disposable and transfer a fixed volume. In some embodiments, the dropper is used to place a sample into the reagent chamber or on the support medium. [428] In some embodiments, the systems described herein comprise at least one of: a detection reagent; and an amplification reagent. In some embodiments, the detection reagent is selected from: a reporter nucleic acid, a detection moiety, and an additional polypeptide, or is a combination thereof; and the amplification reagent is selected from: a primer, a polymerase, a dNTP, and an rNTP, or is a combination thereof. In some embodiments, the detection reagent is operably linked to the polypeptide or the guide nucleic acid, such that a detection event occurs upon contacting the system with a target nucleic acid. In some embodiments, the amplification reagent amplifies a target nucleic acid. [429] In some embodiments, the systems described herein comprise components for modification or detection of a target nucleic acid, wherein the components comprise any one of the guide nucleic acids described herein, any one of the compositions described herein, any one of the nucleic acid expression vectors described herein, or any one of the pharmaceutical compositions described herein. In some embodiments, the systems described herein comprise a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that is at least 90% identical to any one of the sequences recited in TABLE 5. System solutions [430] In general, system components comprise a solution in which the activity of an effector protein occurs. Often, the solution comprises or consists essentially of a buffer. In some embodiments, the solution or buffer comprises a buffering agent, a salt, a crowding agent, a detergent, a reducing agent, a competitor, or a combination thereof. Often the buffer is the primary component or the basis for the solution in which 107 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT the activity occurs. Thus, concentrations for components of buffers described herein (e.g., buffering agents, salts, crowding agents, detergents, reducing agents, and competitors) are the same or essentially the same as the concentration of these components in the solution in which the activity occurs. In some embodiments, a buffer is required for cell lysis activity or viral lysis activity. [431] In some embodiments, systems comprise a buffer, wherein the buffer comprise at least one buffering agent. Exemplary buffering agents include HEPES, TRIS, MES, ADA, PIPES, ACES, MOPSO, BIS-TRIS propane, BES, MOPS, TES, DISO, Trizma, TRICINE, GLY-GLY, HEPPS, BICINE, TAPS, A MPD, A MPSO, CHES, CAPSO, AMP, CAPS, IB1, TCEP, EGTA, Tween 20, KC1, KOH, MgCl2, glycerol, or any combination thereof. In some embodiments, a buffer comprises Tris-HCl pH 8.8, VLB, EGTA, CH3COOH, TCEP, IsoAmp®, (NH4)2SO4, KCl, MgSO4, Tween20, KOAc, MgOAc, BSA, phosphate, citrate, acetate, imidazole, or any combination thereof. In some embodiments, the concentration of the buffering agent in the buffer is 1 mM to 200 mM. In some embodiments, a buffer compatible with an effector protein comprises a buffering agent at a concentration of 10 mM to 30 mM. In some embodiments, a buffer compatible with an effector protein comprises a buffering agent at a concentration of about 20 mM. In some embodiments, a buffering agent provide a pH for the buffer or the solution in which the activity of the effector protein occurs. In some embodiments, the pH is in a range of from 3 to 4, 3.5 to 4.5, 4 to 5, 4.5 to 5.5, 5 to 6, 5.5 to 6.5, 6 to 7, 6.5 to 7.5, 7 to 8, 7.5 to 8.5, 8 to 9, 8.5 to 9.5, 9 to 10, or 9.5 to 10.5. [432] In some embodiments, systems comprise a solution, wherein the solution comprises one or more salt. Accordingly, in some embodiments, the salt is one or more salts selected from a magnesium salt, a zinc salt, a potassium salt, a calcium salt, and a sodium salt. In some embodiments, the salt is a combination of two or more salts. For example, in some embodiments, the salt is a combination of two or more salts selected from a magnesium salt, a zinc salt, a potassium salt, a calcium salt and a sodium salt. In some embodiments, the salt is magnesium acetate. In some embodiments, the salt is magnesium chloride. In some embodiments, the salt is potassium acetate. In some embodiments, the salt is potassium nitrate. In some embodiments, the salt is zinc chloride. In embodiments, the salt is sodium chloride. In some embodiments, the salt is potassium chloride. [433] In some embodiments, the concentration of the one or more salt in the solution is about 0.001 mM to about 500 mM. In some embodiments, the concentration of the salt is about 0.001 mM to about 400 mM. In some embodiments, the concentration of the salt is about 0.001 mM to about 300 mM. In some embodiments, the concentration of the salt is about 0.001 mM to about 200 mM. In some embodiments, the concentration of the salt is about 0.001 mM to about 100 mM. In some embodiments, the concentration of the salt is about 0.001 mM to about 10 mM. In some embodiments, the concentration of the salt is about 0.01 mM to about 500 mM. In some embodiments, the concentration of the salt is about 0.01 mM to about 400 mM. In some embodiments, the concentration of the salt is about 0.01 mM to about 300 mM. In some embodiments, the concentration of the salt is about 0.01 mM to about 200 mM. In some embodiments, the concentration of the salt is about 0.01 mM to about 100 mM. In some embodiments, the concentration of 108 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT the salt is about 0.01 mM to about 10 mM. In some embodiments, the concentration of the salt is about 0.1 mM to about 500 mM. In some embodiments, the concentration of the salt is about 0.1 mM to about 400 mM. In some embodiments, the concentration of the salt is about 0.1 mM to about 300 mM. In some embodiments, the concentration of the salt is about 0.1 mM to about 200 mM. In some embodiments, the concentration of the salt is about 0.1 mM to about 100 mM. In some embodiments, the concentration of the salt is about 0.1 mM to about 10 mM. In some embodiments, the concentration of the salt is about 1 mM to about 500 mM. In some embodiments, the concentration of the salt is about 1 mM to about 400 mM. In some embodiments, the concentration of the salt is about 1 mM to about 300 mM. In some embodiments, the concentration of the salt is about 1 mM to about 200 mM. In some embodiments, the concentration of the salt is about 1 mM to about 100 mM. In some embodiments, the concentration of the salt is about 1 mM to about 10 mM. In some embodiments, the concentration of the salt is about 10 mM to about 500 mM. In some embodiments, the concentration of the salt is about 10 mM to about 400 mM. In some embodiments, the concentration of the salt is about 10 mM to about 300 mM. In some embodiments, the concentration of the salt is about 10 mM to about 200 mM. In some embodiments, the concentration of the salt is about 10 mM to about 100 mM. In some embodiments, the concentration of the salt is about 100 mM to about 500 mM. In some embodiments, the concentration of the salt is about 100 mM to about 400 mM. In some embodiments, the concentration of the salt is about 100 mM to about 300 mM. In some embodiments, the concentration of the salt is about 100 mM to about 200 mM. In some embodiments, the salt is potassium acetate and the concentration of salt in the solution is about 100 mM. In some embodiments, the salt is potassium acetate or sodium chloride and the concentration of salt in the solution is about 200 mM. In some embodiments, the salt is potassium acetate or sodium chloride and the concentration of salt in the solution is about 100 mM to about 200 mM. In some embodiments, the concentration of the at least one salt in the solution is 5 mM to 100 mM, 5 mM to 10 mM, 1 mM to 60 mM, or 1 mM to 10 mM. In some embodiments, the concentration of the at least one salt is about 105 mM. In some embodiments, the concentration of the at least one salt is about 55 mM. In some embodiments, the concentration of the at least one salt is about 7 mM. In some embodiments, the solution comprises potassium acetate and magnesium acetate. In some embodiments, the solution comprises sodium chloride and magnesium chloride. In some embodiments, the solution comprises potassium chloride and magnesium chloride. In some embodiments, the salt is a magnesium salt and the concentration of magnesium in the solution is at least 5 mM, 7 mM, at least 9 mM, at least 11 mM, at least 13 mM, or at least 15 mM. In some embodiments, the concentration of magnesium is less than 20mM, less than 18 mM, or less than 16 mM. [434] In some embodiments, systems comprise a solution, wherein the solution comprises at least one crowding agent. In some embodiments, a crowding agent reduces the volume of solvent available for other molecules in the solution, thereby increasing the effective concentrations of said molecules. Exemplary crowding agents include glycerol and bovine serum albumin. In some embodiments, the crowding agent is glycerol. In some embodiments, the concentration of the crowding agent in the solution is 0.01% (v/v) to 10% (v/v). In some embodiments, the concentration of the crowding agent in the solution is 0.5% (v/v) to 10% (v/v). 109 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [435] In some embodiments, systems comprise a solution, wherein the solution comprises at least one detergent. Exemplary detergents include Tween, Triton-X, and IGEPAL. In some embodiments, a solution comprises Tween, Triton-X, or any combination thereof. In some embodiments, a solution comprises Triton-X. In some embodiments, a solution comprises IGEPAL CA-630. In some embodiments, the concentration of the detergent in the solution is 2% (v/v) or less. In some embodiments, the concentration of the detergent in the solution is 1% (v/v) or less. In some embodiments, the concentration of the detergent in the solution is 0.00001% (v/v) to 0.01% (v/v). In some embodiments, the concentration of the detergent in the solution is about 0.01% (v/v). [436] In some embodiments, systems comprise a solution, wherein the solution comprises at least one reducing agent. Exemplary reducing agents comprise dithiothreitol (DTT), ß-mercaptoethanol (BME), or tris(2-carboxyethyl) phosphine (TCEP). In some embodiments, the reducing agent is DTT. In some embodiments, the concentration of the reducing agent in the solution is 0.01 mM to 100 mM. In some embodiments, the concentration of the reducing agent in the solution is 0.1 mM to 10 mM. In some embodiments, the concentration of the reducing agent in the solution is 0.5 mM to 2 mM. In some embodiments, the concentration of the reducing agent in the solution is 0.01 mM to 100 mM. In some embodiments, the concentration of the reducing agent in the solution is 0.1 mM to 10 mM. In some embodiments, the concentration of the reducing agent in the solution is about 1 mM. [437] In some embodiments, systems comprise a solution, wherein the solution comprises a competitor. In general, competitors compete with the target nucleic acid or the reporter nucleic acid for cleavage by the effector protein or a dimer thereof. Exemplary competitors include heparin, and imidazole, and salmon sperm DNA. In some embodiments, the concentration of the competitor in the solution is 1 µg/mL to 100 µg/mL. In some embodiments, the concentration of the competitor in the solution is 40 µg/mL to 60 µg/mL. [438] In some embodiments, systems comprise a solution, wherein the solution comprises a co-factor. In some embodiments, the co-factor allows an effector protein or a multimeric complex thereof to perform a function, including pre-crRNA processing and/or target nucleic acid cleavage. In some embodiments, the suitability of a cofactor for an effector protein or a multimeric complex thereof is assessed, such as by methods based on those described by Sundaresan et al. (Cell Rep. 2017 Dec 26; 21(13): 3728–3739). In some embodiments, an effector or a multimeric complex thereof forms a complex with a co-factor. In some embodiments, the co-factor is a divalent metal ion. In some embodiments, the divalent metal ion is selected from Mg
2+, Mn
2+, Zn
2+, Ca
2+, Cu
2+. In some embodiments, the divalent metal ion is Mg
2+. In some embodiments, the co-factor is Mg
2+. [439] In some embodiments, systems, and compositions for use with systems comprise a catalytic reagent for signal improvement or enhancement. In some embodiments, the catalytic reagent enhances signal generation via hydrolysis of inorganic pyrophosphates. In some embodiments, catalytic reagents enhance signal generation via enhancement of DNA replication. In some embodiments, catalytic reagents enhance signal amplification via revival of ions (e.g., Mg2+) in a buffer, thereby enhancing the function of an effector protein. In some embodiments, the catalytic reagent for signal improvement comprises an enzyme. 110 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT In some embodiments, the catalytic reagent for signal improvement are particularly useful in amplification and/or detection reactions as described herein. Other exemplary reagents useful for amplification and/or detection reactions (i.e., amplification and detection reagents, respectively) are described throughout herein. [440] Any of the systems, methods, or compositions described herein comprise a catalytic reagent or the use thereof. In some embodiments, compositions comprise about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 enzyme unit (U) of a catalytic reagent per 10 µL of solution. In some embodiments, a catalytic reagent is present in a composition at a concentration of 0.125 Units, 0.5 Units, 0.25 Units, 1.0 Units, 2.0 Units, 2.5 Units, or 4 Units per discrete reaction volume. In some embodiments, a catalytic reagent is provided in a system separately from a buffer provided in the system. In some embodiments, systems comprise a buffer, wherein a catalytic reagent is provided in the buffer. [441] In some embodiments, a catalytic reagent improves the signal to noise ratio of an effector protein- based detection reaction. In some embodiments, a catalytic reagent improves overall signal (e.g., fluorescence of a cleaved reporter). In some embodiments, a catalytic reagent improves signal by a factor, wherein the signal is indicative of the presence of a target nucleic acid. In some embodiments, the factor is at least about 1.1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10. [442] Also provided herein are reagents for: detection reactions, nuclease purification, cell lysis, in vitro transcription reactions, amplification reactions, reverse and transcription reactions. In some embodiments, systems, compositions, and/or solutions described herein comprise one or more of: detection reagents, nuclease purification reagents, cell lysis reagents, in vitro transcription reagents, amplification reagents, reverse transcription reagents, and combinations thereof. In some embodiments, any such reagents suitable with the solutions, compositions, systems, and/or methods described herein are used for achieving one or more of the foregoing described reactions. In some embodiments, reagents provided herein are used with any other solution components described herein, including buffers, amino acids or derivatives thereof, chaotrpes, chelators, cyclodextrins, inhibitors, ionic liquids, linkers, metals, non-detergent sulfobetaines, organic acids, osmolytes, peptides, polyamides, polymers, polyols, polyols and salts, salts, or combinations thereof. Detection Reagents/Components and Reporters [443] In some embodiments, systems disclosed herein comprise detection reagents to facilitate detection of nucleic acids as described herein. Non-limiting examples of detection reagents include a reporter nucleic acid, a detection moiety, and additional polypeptides. In some embodiments, the detection reagent is operably linked to an effector protein described herein such that a detection event occurs upon contacting the detection reagent and effector protein with a target nucleic acid. Upon the occurrence of the detection event, a signal (e.g., a detectable signal or detectable product) can be generated thereby indicating detection of the target nucleic acid. In some embodiments, any suitable detection reagent can be used. Accordingly, 111 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT in some embodiments, the detection reagent comprises a nucleic acid (which, in some embodiments, is referred to herein as a detection or reporter nucleic acid), a detection moiety, an additional polypeptide, or a combination thereof. Other detection reagents include buffers, reverse transcriptase mix, a catalytic reagent, and a stain. Any reagents suitable with the detection reactions, events, and signals described herein are useful as detection reagents for the solutions, compositions, systems, and methods provided herein. In some embodiments, detection reagents are capable of detecting a nucleic acid in a sample. [444] In some embodiments, solutions, compositions, systems, and methods comprise 0.01 µL, 0.02 µL, 0.03 µL, 0.04 µL, 0.05 µL, 0.06 µL, 0.07 µL, 0.08 µL, 0.09 µL, 0.1 µL, 0.2 µL, 0.3 µL, 0.4 µL, 0.5 µL, 0.6 µL, 0.7 µL, 0.8 µL, 0.9 µL, 1 µL, 2 µL, 3 µL, 4 µL, 5 µL, 6 µL, 7 µL, 8 µL, 9 µL, 10 µL, 20 µL, 30 µL, 40 µL, 50 µL, 60 µL, 70 µL, 80 µL, 90 µL, 100 µL, 150 µL, 200 µL, 250 µL, 300 µL, 350 µL, 400 µL, 450 µL, 500 µL, or more of each detection reagent as described herein. In some embodiments, solutions, compositions, systems, and methods comprise 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, or more of each detection reagent as described herein. In some embodiments, solutions, compositions, systems, and methods comprise 1 µM, 2 µM, 3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM, 9 µM, 10 µM, 20 µM, 30 µM, 40 µM, 50 µM, 60 µM, 70 µM, 80 µM, 90 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 350 µM, 400 µM, 450 µM, 500 µM, or more of each detection reagent as described herein. In some embodiments, solutions, compositions, systems, and methods comprise 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, or more of each detection reagent as described herein. [445] In some embodiments, detection reagents are capable of detecting a nucleic acid in a sample. In some embodiments, nucleic acid amplification of the target nucleic acid improves at least one of sensitivity, specificity, or accuracy of the assay in detecting the target nucleic acid. Accordingly, in some embodiments, nucleic acid detection involves PCR or isothermal nucleic acid amplification, providing improved sensitive, specific, or rapid detection. In some embodiments, the reagents or components for nucleic acid detection comprise recombinases, primers, polypeptides, buffers, and signal reagents suitable for a detection reaction. [446] In some embodiments, systems described herein comprise a PCR tube, a PCR well or a PCR plate. In some embodiments, the wells of the PCR plate are pre-aliquoted with the reagent for detecting a nucleic acid, as well as a guide nucleic acid, an effector protein, a multimeric complex, an amplification reagent, or any combination thereof. In some embodiments, the pre-aliquoted guide nucleic acid targeting a target sequence, and an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence. Accordingly, in some embodiments, a user adds a sample of interest to a well of the pre-aliquoted PCR plate. [447] In some embodiments, nucleic acid detection is performed in a nucleic acid detection region on a support medium, or sample interface. Alternatively, or in combination, the nucleic acid detection is 112 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT performed in a reagent chamber, and the resulting sample is applied to the support medium, sample interface, or surface within a reagent chamber. [448] In some embodiments, the reporter nucleic acid is capable of being cleaved by the activated nuclease, thereby generating a detectable signal. Accordingly, in some embodiments, a user adds a sample of interest to a well of the pre-aliquoted PCR plate and measure for the detectable signal with a fluorescent light reader or a visible light reader. [449] In some embodiments, detection reaction of nucleic acid as described herein is performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes, or any value 1 to 60 minutes. In some embodiments, the detection reaction is performed for 1 to 60, 5 to 55, 10 to 50, 15 to 45, 20 to 40, or 25 to 35 minutes. In some embodiments, the detection reaction is performed at a temperature of around 20-45ºC. In some embodiments, the detection reaction is performed at a temperature no greater than 20ºC, 25ºC, 30ºC, 35ºC, 37ºC, 40ºC, 45ºC, or any value 20 ºC to 45 ºC. In some embodiments, the detection reaction is performed at a temperature of at least 20ºC, 25ºC, 30ºC, 35ºC, 37ºC, 40ºC, or 45ºC, or any value 20 ºC to 45 ºC. In some embodiments, the detection reaction is performed at a temperature of 20ºC to 45ºC, 25ºC to 40ºC, 30ºC to 40ºC, or 35ºC to 40ºC. [450] In some embodiments, systems disclosed herein comprise reagents compatible with the samples, solutions, compositions, systems, methods of detection, and support mediums as described herein for detection of an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry. The reagents described herein for detecting a disease, cancer, or genetic disorder comprise a guide nucleic acid targeting the target nucleic acid segment indicative of a disease, cancer, or genetic disorder. [451] In some embodiments, systems disclosed herein comprise a reporter. By way of non-limiting and illustrative example, a reporter comprises a single stranded nucleic acid and a detection moiety (e.g., a labeled single stranded RNA reporter), wherein the nucleic acid is capable of being cleaved by an effector protein (e.g., a protein as disclosed herein) or a multimeric complex thereof, releasing the detection moiety, and generating a detectable signal or a detectable product. In some embodiments, cleavage of the reporter is effective to produce a detectable product comprising a detectable moiety or a detectable signal. The effector proteins disclosed herein, activated upon hybridization of a guide nucleic acid to a target nucleic acid, cleaves the reporter. Cleavage of a reporter produces different types of signals (e.g., a detectable signal). In some embodiments, cleavage of the reporter produces a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal. Various devices and/or sensors can be used to detect these different types of signals, which indicate whether a target nucleic acid, is present in the sample. The sensors usable to detect such signals can include, for example, optical sensors (e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies), electric potential sensors, surface plasmon resonance (SPR) sensors, interferometric sensors, or any other type of sensor suitable for detecting calorimetric signals, potentiometric signals, amperometric signals, optical signals, or piezo-electric signals. 113 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [452] As used herein, a reporter comprises a nucleic acid (e.g., RNA and/or DNA). In some embodiments, a reporter is double-stranded. In some embodiments, a reporter is single-stranded. In some embodiments, a reporter comprise a protein capable of generating a detectable signal or signal. In some embodiments, a reporter is operably linked to the protein capable of generating a signal. In some embodiments, a signal is a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some embodiments, the reporter comprises a detection moiety. In some embodiments, the reporter is configured to release a detection moiety or generate a signal indicative of a presence or absence of the target nucleic acid. For example, the signal can indicate a presence of the target nucleic acid in the sample, and an absence of the signal can indicate an absence of the target nucleic acid in the sample. In some embodiments, suitable detectable labels and/or moieties provide a signal. In some embodiments, non- limiting example of suitable detectable label and/or moiety comprises an enzyme, a radioisotope, a member of a specific binding pair; a fluorophore; a fluorescent protein; and a quantum dot. [453] In some embodiments, the reporter comprises a detection moiety and a quenching moiety. In some embodiments, the reporter comprises a cleavage site, wherein the detection moiety is located at a first site on the reporter and the quenching moiety is located at a second site on the reporter, wherein the first site and the second site are separated by the cleavage site. Sometimes the quenching moiety is a fluorescence quenching moiety. In some embodiments, the quenching moiety is 5’ to the cleavage site and the detection moiety is 3’ to the cleavage site. In some embodiments, the detection moiety is 5’ to the cleavage site and the quenching moiety is 3’ to the cleavage site. Sometimes the quenching moiety is at the 5’ terminus of the nucleic acid of a reporter. Sometimes the detection moiety is at the 3’ terminus of the nucleic acid of a reporter. In some embodiments, the detection moiety is at the 5’ terminus of the nucleic acid of a reporter. In some embodiments, the quenching moiety is at the 3’ terminus of the nucleic acid of a reporter. [454] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin. Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, β- glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, and glucose oxidase (GO). [455] In some embodiments, the detection moiety comprises an invertase. In some embodiments, the substrate of the invertase comprises sucrose. In some embodiments, a DNS reagent that is included in the system for producing a colorimetric change when the invertase converts sucrose to glucose. In some 114 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT embodiments, the reporter nucleic acid and invertase are conjugated using a heterobifunctional linker by sulfo-SMCC chemistry. [456] In some embodiments, suitable fluorophores provide a detectable fluorescence signal in the same range as 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). Non-limiting examples of fluorophores are fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester). In some embodiments, the fluorophore comprises an infrared fluorophore. In some embodiments, the fluorophore emits fluorescence in the range of 500 nm and 720 nm. In some embodiments, the fluorophore emits fluorescence at a wavelength of 700 nm or higher. In other embodiments, the fluorophore emits fluorescence at about 665 nm. In some embodiments, the fluorophore emits fluorescence in the range of 500 nm to 520 nm, 500 nm to 540 nm, 500 nm to 590 nm, 590 nm to 600 nm, 600 nm to 610 nm, 610 nm to 620 nm, 620 nm to 630 nm, 630 nm to 640 nm, 640 nm to 650 nm, 650 nm to 660 nm, 660 nm to 670 nm, 670 nm to 680 nm, 690 nm to 690 nm, 690 nm to 700 nm, 700 nm to 710 nm, 710 nm to 720 nm, or 720 nm to 730 nm. In some embodiments, the fluorophore emits fluorescence in the range 450 nm to 750 nm, 500 nm to 650 nm, or 550 to 650 nm. [457] In some embodiments, systems comprise a quenching moiety. In some embodiments, a quenching moiety is chosen based on its ability to quench the detection moiety. In some embodiments, a quenching moiety comprises a non-fluorescent fluorescence quencher. In some embodiments, a quenching moiety quenches a detection moiety that emits fluorescence in the range of 500 nm and 720 nm. In some embodiments, a quenching moiety quenches a detection moiety that emits fluorescence in the range of 500 nm and 720 nm. In some embodiments, the quenching moiety quenches a detection moiety that emits fluorescence at a wavelength of 700 nm or higher. In other embodiments, the quenching moiety quenches a detection moiety that emits fluorescence at about 660 nm or about 670 nm. In some embodiments, the quenching moiety quenches a detection moiety that emits fluorescence in the range of 500 to 520, 500 to 540, 500 to 590, 590 to 600, 600 to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 690 to 690, 690 to 700, 700 to 710, 710 to 720, or 720 to 730 nm. In some embodiments, the quenching moiety quenches a detection moiety that emits fluorescence in the range 450 nm to 750 nm, 500 nm to 650 nm, or 550 to 650 nm. In some embodiments, a quenching moiety quenches fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester). In some embodiments, a quenching moiety comprises Iowa Black RQ, Iowa Black FQ or IRDye QC-1 Quencher. In some embodiments, a quenching moiety quenches fluorescein amidite, 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). In some embodiments, a quenching moiety comprises Iowa Black RQ (Integrated DNA Technologies), Iowa Black FQ (Integrated DNA Technologies) or IRDye QC-1 Quencher (LiCor). Any of 115 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT the quenching moieties described herein may be from any commercially available source, may be an alternative with a similar function, a generic, or a non-tradename of the quenching moieties listed. [458] In some embodiments, the generation of a detectable product or detectable signal from the release of the detection moiety indicates that cleavage by the effector protein has occurred and that the sample contains the target nucleic acid. In some embodiments, the detection moiety comprises a fluorescent dye. Sometimes the detection moiety comprises a fluorescence resonance energy transfer (FRET) pair. In some embodiments, the detection moiety comprises an infrared (IR) dye. In some embodiments, the detection moiety comprises an ultraviolet (UV) dye. Alternatively, or in combination, the detection moiety comprises a protein. Sometimes the detection moiety comprises an antigen. Sometimes the detection moiety comprises a biotin. Sometimes the detection moiety comprises at least one of avidin or streptavidin. In some embodiments, the detection moiety comprises a polysaccharide, a polymer, or a nanoparticle. In some embodiments, the detection moiety comprises a gold nanoparticle or a latex nanoparticle. [459] In some embodiments, a detection moiety comprises any moiety capable of generating a detectable product or detectable signal upon cleavage of the reporter by the effector protein. In some embodiments, the detectable product comprises a detectable unit generated from the detectable moiety and capable of emitting a detectable signal as described herein. In some embodiments, the detectable product further comprises a detectable label, a fluorophore, a reporter, or a combination thereof. In some embodiments, the detectable product comprises RNA, DNA, or both. In some embodiments, the detectable product is configured to generate a signal indicative of the presence or absence of the target nucleic acid in, for instance, a cell or a sample. [460] In some embodiments, a detection moiety comprises any moiety capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. A nucleic acid of a reporter, sometimes, is protein-nucleic acid that is capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal upon cleavage of the nucleic acid. Often a calorimetric signal is heat produced after cleavage of the nucleic acids of a reporter. Sometimes, a calorimetric signal is heat absorbed after cleavage of the nucleic acids of a reporter. A potentiometric signal, for example, is electrical potential produced after cleavage of the nucleic acids of a reporter. In some embodiments, an amperometric signal comprises movement of electrons produced after the cleavage of nucleic acid of a reporter. Often, the signal is an optical signal, such as a colorimetric signal or a fluorescence signal. An optical signal is, for example, a light output produced after the cleavage of the nucleic acids of a reporter. Sometimes, an optical signal is a change in light absorbance between before and after the cleavage of nucleic acids of a reporter. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the nucleic acid of a reporter. [461] In some embodiments, the detectable signal comprises a colorimetric signal or a signal visible by eye. In some embodiments, the detectable signal can be fluorescent, electrical, chemical, electrochemical, or magnetic. In some embodiments, the first detection signal is generated by interaction of the detection moiety to the capture molecule in the detection region, where the first detection signal indicates that the 116 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT sample contained the target nucleic acid. Sometimes systems are capable of detecting more than one type of target nucleic acid, wherein the system comprises more than one type of guide nucleic acid and more than one type of reporter nucleic acid. In some embodiments, the detectable signal is generated directly by the cleavage event. Alternatively, or in combination, the detectable signal is generated indirectly by the signal event. Sometimes the detectable signal is not a fluorescent signal. In some embodiments, the detectable signal comprises a colorimetric or color-based signal. In some embodiments, the detected target nucleic acid is identified based on its spatial location on the detection region of the support medium. In some embodiments, the second detectable signal is generated in a spatially distinct location than the first generated signal. [462] In some embodiments, the reporter nucleic acid is a single-stranded nucleic acid sequence comprising ribonucleotides. In some embodiments, the nucleic acid of a reporter comprises a single- stranded nucleic acid sequence comprising at least one ribonucleotide. In some embodiments, the nucleic acid of a reporter is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some embodiments, the nucleic acid of a reporter comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 ribonucleotide residues at an internal position. In some embodiments, the nucleic acid of a reporter comprises from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between non-ribonucleotide residues. In some embodiments, the nucleic acid of a reporter has only ribonucleotide residues. In some embodiments, the nucleic acid of a reporter has only DNA residues. In some embodiments, the nucleic acid comprises nucleotides resistant to cleavage by the effector protein described herein. In some embodiments, the nucleic acid of a reporter comprises synthetic nucleotides. In some embodiments, the nucleic acid of a reporter comprises at least one ribonucleotide residue and at least one non-ribonucleotide residue. [463] In some embodiments, the nucleic acid of a reporter comprises at least one uracil ribonucleotide. In some embodiments, the nucleic acid of a reporter comprises at least two uracil ribonucleotides. Sometimes the nucleic acid of a reporter has only uracil ribonucleotides. In some embodiments, the nucleic acid of a reporter comprises at least one adenine ribonucleotide. In some embodiments, the nucleic acid of a reporter comprises at least two adenine ribonucleotides. In some embodiments, the nucleic acid of a reporter has only adenine ribonucleotides. In some embodiments, the nucleic acid of a reporter comprises at least one cytosine ribonucleotide. In some embodiments, the nucleic acid of a reporter comprises at least two cytosine ribonucleotides. In some embodiments, the nucleic acid of a reporter comprises at least one guanine ribonucleotide. In some embodiments, the nucleic acid of a reporter comprises at least two guanine ribonucleotides. In some embodiments, a nucleic acid of a reporter comprises a single unmodified ribonucleotide. In some embodiments, a nucleic acid of a reporter comprises only unmodified DNAs. [464] In some embodiments, the nucleic acid of a reporter is 5 to 20, 5 to 15, 5 to 10, 7 to 20, 7 to 15, or 7 to 10 nucleotides in length. In some embodiments, the nucleic acid of a reporter is 3 to 20, 4 to 10, 5 to 117 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT 10, or 5 to 8 nucleotides in length. In some embodiments, the nucleic acid of a reporter is 5 to 12 nucleotides in length. In some embodiments, the reporter nucleic acid is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 nucleotides in length. In some embodiments, the reporter nucleic acid is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. [465] In some embodiments, systems comprise a plurality of reporters. In some embodiments, the plurality of reporters comprise a plurality of signals. In some embodiments, systems comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, or at least 50 reporters. In some embodiments, there are 2 to 50, 3 to 40, 4 to 30, 5 to 20, or 6 to 10 different reporters. [466] In some embodiments, systems comprise an effector protein and a reporter nucleic acid configured to undergo trans cleavage by the effector protein. In some embodiments, Trans cleavage of the reporter generates a signal from the reporter or alter a signal from the reporter. In some embodiments, the signal is an optical signal, such as a fluorescence signal or absorbance band. In some embodiments, Trans cleavage of the reporter alters the wavelength, intensity, or polarization of the optical signal. For example, in some embodiments, the reporter comprises a fluorophore and a quencher, such that trans cleavage of the reporter separates the fluorophore and the quencher thereby increasing a fluorescence signal from the fluorophore. Herein, in some embodiments, detection of reporter cleavage to determine the presence of a target nucleic acid is referred to as ‘DETECTR’. In some embodiments described herein is a method of assaying for a target nucleic acid in a sample comprising contacting the target nucleic acid with an effector protein, a non- naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, and a reporter nucleic acid, and assaying for a change in a signal, wherein the change in the signal is produced by cleavage of the reporter nucleic acid. [467] In the presence of a large amount of non-target nucleic acids, in some embodiments, an activity of an effector protein (e.g., an effector protein as disclosed herein) is inhibited. This is because the activated effector proteins collaterally cleave any nucleic acids. In some embodiments, if total nucleic acids are present in large amounts, they outcompete reporters for the effector proteins. In some embodiments, systems comprise an excess of reporter(s), such that when the system is operated and a solution of the system comprising the reporter is combined with a sample comprising a target nucleic acid, the concentration of the reporter in the combined solution-sample is greater than the concentration of the target nucleic acid. In some embodiments, the sample comprises amplified target nucleic acid. In some embodiments, the sample comprises an unamplified target nucleic acid. In some embodiments, the concentration of the reporter is greater than the concentration of target nucleic acids and non-target nucleic acids. In some embodiments, the non-target nucleic acids from the original sample, either lysed or unlysed. In some embodiments, the non-target nucleic acids comprise byproducts of amplification. In some 118 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT embodiments, systems comprise a reporter wherein the concentration of the reporter in a solution 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold excess of total nucleic acids. Amplification Reagents/Components [468] In some embodiments, systems described herein comprise a reagent or component for amplifying a nucleic acid. Non-limiting examples of reagents for amplifying a nucleic acid include polymerases, primers, and nucleotides. In some embodiments, systems comprise reagents for nucleic acid amplification of a target nucleic acid in a sample. In some embodiments, nucleic acid amplification of the target nucleic acid improves at least one of sensitivity, specificity, or accuracy of the assay in detecting the target nucleic acid. In some embodiments, nucleic acid amplification is isothermal nucleic acid amplification, providing for the use of the system or system in remote regions or low resource settings without specialized equipment for amplification. In some embodiments, amplification of the target nucleic acid increases the concentration of the target nucleic acid in the sample relative to the concentration of nucleic acids that do not correspond to the target nucleic acid. [469] In some embodiments, the reagents for nucleic acid amplification comprise a recombinase, a primer, an oligonucleotide primer, an activator, a deoxynucleoside triphosphate (dNTP), a ribonucleoside tri-phosphate (rNTP), a single-stranded DNA binding (SSB) protein, Rnase inhibitor, water, a polymerase, reverse transcriptase mix, or a combination thereof that is suitable for an amplification reaction. Non- limiting examples of amplification reactions are transcription mediated amplification (TMA), helicase dependent amplification (HDA), or circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge- initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), and improved multiple displacement amplification (IMDA). [470] Such amplification reactions, in some embodiments, are also used in combination with reverse transcription (RT) of an RNA of interest. Accordingly, also provided herein are reagents for both the reverse transcription and amplification of nucleic acids. In some embodiments, solutions, compositions, systems and methods comprise 0.01 µL, 0.02 µL, 0.03 µL, 0.04 µL, 0.05 µL, 0.06 µL, 0.07 µL, 0.08 µL, 0.09 µL, 0.1 µL, 0.2 µL, 0.3 µL, 0.4 µL, 0.5 µL, 0.6 µL, 0.7 µL, 0.8 µL, 0.9 µL, 1 µL, 2 µL, 3 µL, 4 µL, 5 µL, 6 µL, 7 µL, 8 µL, 9 µL, 10 µL, 20 µL, 30 µL, 40 µL, 50 µL, 60 µL, 70 µL, 80 µL, 90 µL, 100 µL, 150 µL, 200 µL, 250 µL, 300 µL, 350 µL, 400 µL, 450 µL, 500 µL, or more of each amplification described herein. In 119 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT some embodiments, solutions, compositions, systems and methods comprise 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, or more of each amplification reagent as described herein. In some embodiments, solutions, compositions, systems and methods comprise 1 µM, 2 µM, 3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM, 9 µM, 10 µM, 20 µM, 30 µM, 40 µM, 50 µM, 60 µM, 70 µM, 80 µM, 90 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 350 µM, 400 µM, 450 µM, 500 µM, or more of each amplification reagent as described herein. In some embodiments, solutions, compositions, systems and methods comprise 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, or more of each amplification reagent as described herein. [471] In some embodiments, systems described herein comprise a PCR tube, a PCR well or a PCR plate. In some embodiments, the wells of the PCR plate are pre-aliquoted with the reagent for amplifying a nucleic acid, as well as a guide nucleic acid, an effector protein, a multimeric complex, or any combination thereof. In some embodiments, the wells of the PCR plate are pre-aliquoted with a guide nucleic acid targeting a target sequence, an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence, an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence, and at least one population of a single stranded reporter nucleic acid comprising a detection moiety. In some embodiments, a user thus adds the biological sample of interest to a well of the pre-aliquoted PCR plate and measure for the detectable signal with a fluorescent light reader or a visible light reader. [472] In some embodiments, systems comprise a PCR plate; a guide nucleic acid targeting a target sequence; an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence; and a single stranded reporter nucleic acid comprising a detection moiety, wherein the reporter nucleic acid is capable of being cleaved by the activated nuclease, thereby generating a detectable signal. [473] In some embodiments, systems described herein comprise a support medium; a guide nucleic acid targeting a target sequence; and an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence. In some embodiments, nucleic acid amplification is performed in a nucleic acid amplification region on the support medium. Alternatively, or in combination, the nucleic acid amplification is performed in a reagent chamber, and the resulting sample is applied to the support medium. [474] In some embodiments, a system described herein for editing a target nucleic acid comprises a PCR plate; a guide nucleic acid targeting a target sequence; and an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence. In some embodiments, the wells of the PCR plate are pre-aliquoted with the guide nucleic acid targeting a target sequence, and an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence. In 120 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT some embodiments, a user thus adds the biological sample of interest to a well of the pre-aliquoted PCR plate. [475] Often, the nucleic acid amplification is performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes, or any value 1 to 60 minutes. In some embodiments, the amplification reaction is performed for 1 to 60, 5 to 55, 10 to 50, 15 to 45, 20 to 40, or 25 to 35 minutes. In some embodiments, the amplification reaction is performed at a temperature of around 20-45ºC. In some embodiments, the amplification reaction is performed at a temperature no greater than 20ºC, 25ºC, 27ºC, 30ºC, 35ºC, 37ºC, 40ºC, 45ºC, 47ºC, 50ºC, 55ºC, 57ºC, 60ºC, 65ºC, 67ºC, 70ºC, 75ºC, 77ºC, 80ºC, or any value 20 ºC to 80 ºC. In some embodiments, the amplification reaction is performed at a temperature of at least 20ºC, 25ºC, 27ºC, 30ºC, 35ºC, 37ºC, 40ºC, 45ºC, 47ºC, 50ºC, 55ºC, 57ºC, 60ºC, 65ºC, 67ºC, 70ºC, 75ºC, 77ºC, 80ºC or any value 20 ºC to 80 ºC. In some embodiments, the amplification reaction is performed at a temperature of 20ºC to 45ºC, 25ºC to 40ºC, 30ºC to 40ºC, 35ºC to 40ºC, 40ºC to 45ºC, 45ºC to 50ºC, 50ºC to 55ºC, 55ºC to 60ºC, 35ºC to 40ºC, 50ºC to 65ºC, 65ºC to 70ºC, 70ºC to 80ºC, 75ºC to 80ºC. [476] In some embodiments, systems comprise primers for amplifying a target nucleic acid to produce an amplification product comprising the target nucleic acid and a PAM. In some embodiments, at least one of the primers comprise the PAM that is incorporated into the amplification product during amplification. The compositions for amplification of target nucleic acids and methods of use thereof, as described herein, are compatible with any of the methods disclosed herein including methods of assaying for at least one base difference (e.g., assaying for a SNP or a base mutation) in a target nucleic acid, methods of assaying for a target nucleic acid that lacks a PAM by amplifying the target nucleic acid to introduce a PAM, and compositions used in introducing a PAM by amplification into the target nucleic acid. Additional System Components [477] In some embodiments, systems include a package, carrier, or container that is compartmentalized to receive one or more containers such as vials, or tubes, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, test wells, bottles, vials, syringes, and test tubes. In some embodiments, the containers are formed from a variety of materials such as glass, plastic, or polymers. In some embodiments, the system or systems described herein contain packaging materials. Examples of packaging materials include, but are not limited to, pouches, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for intended mode of use. [478] In some embodiments, systems described herein include labels listing contents and/or instructions for use, or package inserts with instructions for use. In some embodiments, the systems include a set of instructions and/or a label is on or associated with the container. In some embodiments, the label is on a container when letters, numbers or other characters forming the label are attached, molded, or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier 121 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT that also holds the container (e.g., as a package insert). In some embodiments, the label is used to indicate that the contents are to be used for a specific therapeutic application. In some embodiments, the label indicates directions for use of the contents, such as in the methods described herein. In some embodiments, after packaging the formed product and wrapping or boxing to maintain a sterile barrier, the product is terminally sterilized by heat sterilization, gas sterilization, gamma irradiation, or by electron beam sterilization. Alternatively, in some embodiments, the product is prepared and packaged by aseptic processing. [479] In some embodiments, systems comprise a solid support. In some embodiments, an RNP or effector protein is attached to a solid support. In some embodiments, the solid support comprises an electrode or a bead. In some embodiments, the bead comprises a magnetic bead. Upon cleavage, the RNP is liberated from the solid support and interacts with other mixtures. For example, upon cleavage of the nucleic acid of the RNP, the effector protein of the RNP flows through a chamber into a mixture comprising a substrate. When the effector protein meets the substrate, a reaction occurs, such as a colorimetric reaction, which is then detected. As another example, the protein is an enzyme substrate, and upon cleavage of the nucleic acid of the enzyme substrate-nucleic acid, the enzyme flows through a chamber into a mixture comprising the enzyme. When the enzyme substrate meets the enzyme, a reaction occurs, such as a calorimetric reaction, which is then detected. Certain System Conditions [480] In some embodiments, systems and methods are employed under certain conditions that enhance an activity of the effector protein relative to alternative conditions, as measured by a detectable signal released from cleavage of a reporter in the presence of the target nucleic acid. In some embodiments, the detectable signal is generated at about the rate of trans cleavage of a reporter nucleic acid. In some embodiments, the reporter nucleic acid is a homopolymeric reporter nucleic acid comprising 5 to 20 consecutive adenines (SEQ ID NO: 169), 5 to 20 consecutive thymines (SEQ ID NO: 170), 5 to 20 consecutive cytosines (SEQ ID NO: 171), or 5 to 20 consecutive guanines (SEQ ID NO: 172). In some embodiments, the reporter is an RNA-FQ reporter. [481] In some embodiments, effector proteins disclosed herein recognize, bind, or are activated by, different target nucleic acids having different sequences, but are active toward the same reporter nucleic acid, allowing for facile multiplexing in a single assay having a single ssRNA-FQ reporter. [482] In some embodiments, systems are employed under certain conditions that enhance trans cleavage activity of an effector protein. In some embodiments, under certain conditions, trans cleavage occurs at a rate of at least 0.005 mmol/min, at least 0.01 mmol/min, at least 0.05 mmol/min, at least 0.1 mmol/min, at least 0.2 mmol/min, at least 0.5 mmol/min, or at least 1 mmol/min. In some embodiments, systems and methods are employed under certain conditions that enhance cis cleavage activity of the effector protein. 122 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [483] Certain conditions that may enhance the activity of an effector protein include a certain salt presence or salt concentration of the solution in which the activity occurs. For example, in some embodiments, cis cleavage activity of an effector protein is inhibited or halted by a high salt concentration. In some embodiments, the salt comprises a magnesium salt, a zinc salt, a potassium salt, a calcium salt, a lithium salt, an ammonium salt, or a sodium salt. In some embodiments, the salt is magnesium acetate. In some embodiments, the salt is magnesium chloride. In some embodiments, the salt is potassium acetate. In some embodiments, the salt is potassium nitrate. In some embodiments, the salt is zinc chloride. In embodiments, the salt is sodium chloride. In some embodiments, the salt is potassium chloride. In some embodiments, the salt is lithium acetate. In some embodiments, the salt is ammonium sulfate. In some embodiments, the salt concentration is less than 150 mM, less than 125 mM, less than 100 mM, less than 75 mM, less than 50 mM, or less than 25 mM. In some embodiments, the salt concentration is more than 1 mM, but less than 150 mM, less than 125 mM, less than 100 mM, less than 75 mM, less than 50 mM, or less than 25 mM. In some embodiments, the salt concentration is more than 10 mM, but less than 150 mM, less than 125 mM, less than 100 mM, less than 75 mM, less than 50 mM, or less than 25 mM. In some embodiments, the salt is potassium acetate or sodium chloride and the concentration of salt in the solution is about 200 mM. In some embodiments, the salt is potassium acetate or, sodium chloride, lithium acetate, or ammonium sulfate and the concentration of salt in the solution is about 100 mM to about 200 mM. [484] Certain conditions that may enhance the activity of an effector protein include the pH of a solution in which the activity. For example, in some embodiments, increasing pH enhances trans cleavage activity. For example, in some embodiments, the rate of trans cleavage activity increases with increase in pH up to pH 9. In some embodiments, the pH is about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9. In some embodiments, the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5. In some embodiments, the pH is less than 7. In some embodiments, the pH is greater than 7. [485] Certain conditions that may enhance the activity of an effector protein includes the temperature at which the activity is performed. In some embodiments, the temperature is about 25ºC to about 80ºC. In some embodiments, the temperature is about 20°C to about 40°C, about 30°C to about 50°C, or about 40°C to about 60°C. In some embodiments, the temperature is about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55ºC, about 60ºC, about 65°C, about 70°C, about 75°C, or about 80°C. Methods and Formulations for Introducing System Components and Compositions into a Target Cell [486] Disclosed herein, in some aspects, are systems and methods for introducing systems and components of such systems into a target cell. In some embodiments, the systems comprise, as described herein, one or more components having any one of the polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) or a nucleic acid comprising a nucleotide sequence 123 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT encoding same. In some embodiments, such systems comprise, as described herein, one or more components having a guide nucleic acid or a nucleic acid comprising a nucleotide sequence encoding same. In some embodiments, systems comprise one or more components having a guide nucleic acid and an additional nucleic acid. In some embodiments, systems and components thereof are used to introduce the polypeptides, guide nucleic acids, or combinations thereof into a target cell. In some embodiments, the methods are used for modifying or editing a target nucleic acid. In some embodiments, systems comprise the polypeptide, one or more guide nucleic acids, and a reagent for facilitating the introduction of the polypeptide and the one or more guide nucleic acids. In some embodiments, system components for the methods comprise a solution, a buffer, a reagent for facilitating the introduction of the polypeptide and the one or more guide nucleic acids, or combinations thereof. In some embodiments, a guide nucleic acid (or a nucleic acid comprising a nucleotide sequence encoding same) and/or a polypeptide (e.g., effector protein, effector partner, fusion protein, or combination thereof) (or a nucleic acid comprising a nucleotide sequence encoding same) described herein are introduced into a host cell by any of a variety of well-known methods. As a non-limiting example, in some embodiments, the guide nucleic acid and/or polypeptide are combined with a lipid. As another non-limiting example, in some embodiments, the guide nucleic acid and/or polypeptide are combined with a particle or formulated into a particle. Methods for Introducing System Components and Compositions to a Host [487] Described herein are methods of introducing various components described herein to a host. A host may be any suitable host. In some embodiments, a host comprises a host cell. When described herein, a host cell comprises an in vivo or in vitro eukaryotic cell, a prokaryotic cell (e.g., bacterial or archaeal cell), or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity. In some embodiments, eukaryotic or prokaryotic cells are, or have been, used as recipients for methods of introduction described herein. In some embodiments, eukaryotic or prokaryotic cells comprise the progeny of the original cell which has been transformed by the methods of introduction described herein. It is understood that the progeny of a single cell is not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. In some embodiments, a host cell comprises a recombinant host cell or a genetically modified host cell, if a heterologous nucleic acid, e.g., an expression vector, has been introduced into the cell. [488] Methods of introducing a nucleic acid and/or protein into a host cell are known in the art, and any convenient method may be used to introduce a subject nucleic acid (e.g., an expression construct/vector) into a target cell (e.g., a human cell). Suitable methods include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, and nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al. Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023). In some embodiments, the nucleic acid 124 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT and/or protein(s) are introduced into a disease cell comprised in a pharmaceutical composition comprising the guide nucleic acid, the polypeptide, a pharmaceutically acceptable excipient, or combinations thereof. [489] In some embodiments, molecules of interest, such as nucleic acids of interest, are introduced to a host. In some embodiments, polypeptides are introduced to a host. In some embodiments, vectors, such as lipid particles and/or viral vectors are introduced to a host. In some embodiments, introduction is for contact with a host or for assimilation into the host, for example, introduction into a host cell. [490] In some embodiments, described herein are methods of introducing one or more nucleic acids, such as a nucleic acid encoding a polypeptide, a nucleic acid that, when transcribed, produces an engineered guide nucleic acid, or combinations thereof, into a host cell. Any suitable method may be used to introduce a nucleic acid into a cell. Suitable methods include, for example, viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE- dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery. Further methods are described throughout. [491] In some embodiments, introducing one or more nucleic acids into a host cell occurs in any culture media and under any culture conditions that promote the survival of the cells. In some embodiments, introducing one or more nucleic acids into a host cell is carried out in vivo or ex vivo. In some embodiments, introducing one or more nucleic acids into a host cell is carried out in vitro. [492] In some embodiments, polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) are provided as RNA. In some embodiments, the RNA is provided by direct chemical synthesis or is transcribed in vitro from a DNA (e.g., encoding the polypeptide). Once synthesized, the RNA is introduced into a cell by way of any suitable technique for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.). In some embodiments, introduction of one or more nucleic acid is through the use of a vector and/or a vector system, accordingly, in some embodiments, compositions and system described herein comprise a vector and/or a vector system. [493] In some embodiments, vectors are introduced directly to a host. In some embodiments, host cells are contacted with one or more vectors as described herein, and in some embodiments, said vectors are taken up by the cells. Methods for contacting cells with vectors include but are not limited to electroporation, calcium chloride transfection, microinjection, lipofection, micro-injection, contact with the cell or particle that comprises a molecule of interest, or a package of cells or particles that comprise molecules of interest. [494] In some embodiments, components described herein are introduced directly to a host. For example, in some embodiments, an engineered guide nucleic acid is introduced to a host, specifically introduced into a host cell. Methods of introducing nucleic acids, such as RNA into cells include, but are not limited to direct injection, transfection, or any other method used for the introduction of nucleic acids. 125 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [495] In some embodiments, polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) described herein are introduced directly to a host. In some embodiments, polypeptides described herein are modified to promote introduction to a host. For example, in some embodiments, polypeptides described herein are modified to increase the solubility of the polypeptide. In some embodiments, the polypeptide is optionally fused to a polypeptide domain that increases solubility. In some embodiments, the domain is linked to the polypeptide through a defined protease cleavage site, such as TEV sequence which is cleaved by TEV protease. In some embodiments, the linker comprises one or more flexible sequences, e.g., from 1 to 10 glycine residues. In some embodiments, the cleavage of the polypeptide is performed in a buffer that maintains solubility of the product, e.g., in the presence of from 0.5 to 2 M urea, or in the presence of polypeptides and/or polynucleotides that increase solubility. Domains of interest include endosomolytic domains, e.g., influenza HA domain; and other polypeptides that aid in production, e.g., IF2 domain, GST domain, and GRPE domain. In another example, the polypeptide is modified to improve stability. For example, the polypeptides is PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream. In some embodiments, polypeptides are modified to promote uptake by a host, such as a host cell. For example, a polypeptide described herein is fused to a polypeptide permeant domain to promote uptake by a host cell. Any suitable permeant domains may be used in the non-integrating polypeptides of the present disclosure, including peptides, peptidomimetics, and non-peptide carriers. Examples include penetratin, a permeant peptide that is derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia; the HIV-1 tat basic region amino acid sequence, e.g., amino acids 49-57 of a naturally-occurring tat protein; and poly-arginine motifs, for example, the region of amino acids 34-56 of HIV-1 rev protein, nonaarginine, and octa-arginine. In some embodiments, the site at which the fusion is made is selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide. In some embodiments, the optimal site is determined by suitable methods. Formulations for Introducing System Components and Compositions to a Host [496] Described herein are formulations of introducing compositions or components of a system described herein to a host. In some embodiments, such formulations, systems and compositions described herein comprise polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) and a carrier (e.g., excipient, diluent, vehicle, or filling agent). In some aspects of the present disclosure, the polypeptides are provided in a pharmaceutical composition comprising the polypeptides and any pharmaceutically acceptable excipient, carrier, or diluent. Methods of Modifying a Nucleic Acid [497] Provided herein are compositions, methods, and systems for modifying (e.g., editing) target nucleic acids. In general, modifying refers to changing the physical composition of a target nucleic acid. However, compositions, methods, and systems disclosed herein are capable of modifying target nucleic acids, such as making epigenetic modifications of target nucleic acids, which does not change the nucleotide sequence 126 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT of the target nucleic acids per se. In some embodiments, polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof), compositions and systems described herein are used for modifying a target nucleic acid, which includes editing a target nucleic acid sequence. In some embodiments, modifying a target nucleic acid comprises one or more of: cleaving the target nucleic acid, deleting one or more nucleotides of the target nucleic acid, inserting one or more nucleotides into the target nucleic acid, mutating one or more nucleotides of the target nucleic acid, or otherwise changing one or more nucleotides of the target nucleic acid. In some embodiments, modifying a target nucleic acid comprises one or more of: methylating, demethylating, deaminating, or oxidizing one or more nucleotides of the target nucleic acid. [498] In some embodiments, compositions, methods, and systems described herein modify a coding portion of a gene, a non-coding portion of a gene, or a combination thereof. In some embodiments, modifying at least one gene using the compositions, methods or systems described herein reduce or increase expression of one or more genes. In some embodiments, the compositions, methods or systems reduce expression of one or more genes by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the compositions, methods or systems remove all expression of a gene, also referred to as genetic knock out. In some embodiments, the compositions, methods or systems increase expression of one or more genes by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. [499] In some embodiments, the compositions, methods or systems comprise a nucleic acid expression vector, or use thereof, to introduce polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof), guide nucleic acid, donor template or any combination thereof to a cell. In some embodiments, the nucleic acid expression vector is a viral vector. Viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses. In some embodiments, the viral vector is a replication-defective viral vector, comprising an insertion of a therapeutic gene inserted in genes essential to the lytic cycle, preventing the virus from replicating and exerting cytotoxic effects. In some embodiments, the viral vector is an adeno associated viral (AAV) vector. In some embodiments, the nucleic acid expression vector is a non-viral vector. In some embodiments, compositions and methods comprise a lipid, polymer, nanoparticle, or a combination thereof, or use thereof, to introduce the polypeptide, guide nucleic acid, donor template or any combination thereof to a cell. Non-limiting examples of lipids and polymers are cationic polymers, cationic lipids, or bio-responsive polymers. In some embodiments, the bio-responsive polymer exploits chemical-physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space. [500] In some embodiments, methods of modifying comprise contacting a target nucleic acid with one or more components, compositions or systems described herein. In some embodiments, a method of modifying comprises contacting a target nucleic acid with at least one of: a) one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof), or one or more nucleic acids 127 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT encoding the one or more polypeptides; or b) one or more guide nucleic acids, or one or more nucleic acids encoding one or more guide nucleic acids. In some embodiments, a method of modifying comprises contacting a target nucleic acid with a system described herein wherein the system comprises components comprising at least one of: a) one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof), or one or more nucleic acids encoding the one or more polypeptides; or b) one or more guide nucleic acids, or one or more nucleic acids encoding one or more guide nucleic acids. In some embodiments, a method of modifying comprises contacting a target nucleic acid with a composition described herein comprising at least one of: a) one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof), or one or more nucleic acids encoding the one or more polypeptides; or b) one or more guide nucleic acids, or one or more nucleic acids encoding one or more guide nucleic acids; in a composition. In some embodiments, a method of modifying as described herein produces a modified target nucleic acid. [501] In some embodiments, editing a target nucleic acid sequence introduces a mutation (e.g., point mutations, deletions) in a target nucleic acid relative to a corresponding wildtype nucleotide sequence. In some embodiments, editing removes or corrects a disease-causing mutation in a nucleic acid sequence to produce a corresponding wildtype nucleotide sequence. In some embodiments, editing a target nucleic acid sequence removes/corrects point mutations, deletions, null mutations, or tissue-specific mutations in a target nucleic acid. In some embodiments, editing a target nucleic acid sequence is used for generating gene knock-out, gene knock-in, gene editing, gene tagging, or a combination thereof. In some embodiments, methods of the disclosure are targeted to any locus in a genome of a cell. [502] In some embodiments, modifying comprises single stranded cleavage, double stranded cleavage, donor nucleic acid insertion, epigenetic modification (e.g., methylation, demethylation, acetylation, or deacetylation), or a combination thereof. In some embodiments, cleavage (single-stranded or double- stranded) is site-specific, meaning cleavage occurs at a specific site in the target nucleic acid, often within the region of the target nucleic acid that hybridizes with the guide nucleic acid spacer sequence. In some embodiments, the polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof)introduce a single-stranded break in a target nucleic acid to produce a cleaved nucleic acid. In some embodiments, the polypeptide is capable of introducing a break in a single stranded RNA (ssRNA). In some embodiments, the polypeptide is coupled to a guide nucleic acid that targets a particular region of interest in the ssRNA. In some embodiments, the target nucleic acid, and the resulting cleaved nucleic acid is contacted with a nucleic acid for homologous recombination (e.g., homology directed repair (HDR)) or non-homologous end joining (NHEJ). In some embodiments, a double-stranded break in the target nucleic acid is repaired (e.g., by NHEJ or HDR) without insertion of a donor template, such that the repair results in an indel in the target nucleic acid at or near the site of the double-stranded break. In some embodiments, an indel, sometimes referred to as an insertion-deletion or indel mutation, is a type of genetic mutation that results from the insertion and/or deletion of one or more nucleotide in a target nucleic acid. In some embodiments, an indel varies in length (e.g., 1 to 1,000 nucleotides in length) and be detected using methods 128 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT well known in the art, including sequencing. If the number of nucleotides in the insertion/deletion is not divisible by three, and it occurs in a protein coding region, it is also a frameshift mutation. Indel percentage is the percentage of sequencing reads that show at least one nucleotide has been mutation that results from the insertion and/or deletion of nucleotides regardless of the size of insertion or deletion, or number of nucleotides mutated. For example, if there is at least one nucleotide deletion detected in a given target nucleic acid, it counts towards the percent indel value. As another example, if one copy of the target nucleic acid has one nucleotide deleted, and another copy of the target nucleic acid has 10 nucleotides deleted, they are counted the same. This number reflects the percentage of target nucleic acids that are edited by a given polypeptide. [503] In some embodiments, methods of modifying described herein cleave a target nucleic acid at one or more locations to generate a cleaved target nucleic acid. In some embodiments, the cleaved target nucleic acid undergoes recombination (e.g., NHEJ or HDR). In some embodiments, cleavage in the target nucleic acid is repaired (e.g., by NHEJ or HDR) without insertion of a donor nucleic acid, such that the repair results in an indel in the target nucleic acid at or near the site of the cleavage site. In some embodiments, cleavage in the target nucleic acid is repaired (e.g., by NHEJ or HDR) with insertion of a donor nucleic acid, such that the repair results in an indel in the target nucleic acid at or near the site of the cleavage site. [504] In some embodiments, wherein the compositions, systems, and methods of the present disclosure restore a wild-type reading frame. In some embodiments, a wild-type reading frame comprises a reading frame that produces at least a partially, or fully, functional protein. In some embodiments, a non-wild-type reading frame comprises a reading frame that produces a non-functional or partially non-functional protein. [505] Accordingly, in some embodiments, compositions, systems, and methods described herein edit 1 to 1,000 nucleotides or any integer in between, in a target nucleic acid. In some embodiments, 1 to 1,000, 2 to 900, 3 to 800, 4 to 700, 5 to 600, 6 to 500, 7 to 400, 8 to 300, 9 to 200, or 10 to 100 nucleotides, or any integer in between, are edited by the compositions, systems, and methods described herein. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides are edited by the compositions, systems, and methods described herein. In some embodiments, 10, 20, 30, 40, 50, 60, 70, 8090, 100 or more nucleotides, or any integer in between, are edited by the compositions, systems, and methods described herein. In some embodiments, 100, 200, 300, 400, 500, 600, 700, 800, 900 or more nucleotides, or any integer in between, are edited by the compositions, systems, and methods described herein. [506] In some embodiments, methods comprise use of two or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof). An illustrative method for introducing a break in a target nucleic acid comprises contacting the target nucleic acid with: (a) a first engineered guide nucleic acid comprising a region that binds to a first polypeptide described herein; and (b) a second engineered guide nucleic acid comprising a region that binds to a second polypeptide described herein, wherein the first engineered guide nucleic acid comprises an additional region that hybridizes to the target nucleic acid and wherein the second engineered guide nucleic acid comprises an additional region that hybridizes to the 129 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT target nucleic acid. In some embodiments, the first and second polypeptide are identical. In some embodiments, the first and second polypeptide are not identical. [507] In some embodiments, editing a target nucleic acid comprises genome editing. In some embodiments, genome editing comprises editing a genome, chromosome, plasmid, or other genetic material of a cell or organism. In some embodiments, the genome, chromosome, plasmid, or other genetic material of the cell or organism is modified in vivo. In some embodiments, the genome, chromosome, plasmid, or other genetic material of the cell or organism is modified in a cell. In some embodiments, the genome, chromosome, plasmid, or other genetic material of the cell or organism is modified in vitro. For example, in some embodiments, a plasmid is edited in vitro using a composition described herein and introduced into a cell or organism. [508] In some embodiments, editing a target nucleic acid comprises deleting a sequence from a target nucleic acid. For example, in some embodiments, a mutated sequence or a sequence associated with a disease is removed from a target nucleic acid. In some embodiments, editing a target nucleic acid comprises replacing a sequence in a target nucleic acid with a second sequence. For example, in some embodiments, a mutated sequence or a sequence associated with a disease is replaced with a second sequence lacking the mutation or that is not associated with the disease. In some embodiments, editing a target nucleic acid comprises deleting or replacing a sequence comprising markers associated with a disease or disorder. In some embodiments, editing a target nucleic acid comprises introducing a sequence into a target nucleic acid. For example, in some embodiments, a beneficial sequence or a sequence that reduces or eliminates a disease is inserted into the target nucleic acid. [509] In some embodiments, methods comprise inserting a donor nucleic acid into a cleaved target nucleic acid. In some embodiments, the donor nucleic acid is inserted at a specified (e.g., effector protein targeted) point within the target nucleic acid. In some embodiments, the cleaved target nucleic acid is cleaved at a single location. In such embodiments, the methods comprise contacting a target nucleic acid with an effector protein described herein, thereby introducing a single-stranded break in the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally by HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., at a cleavage site). In some embodiments, the cleaved target nucleic acid is cleaved at two locations. In such embodiments, the methods comprise contacting a target nucleic acid with an effector protein described herein, thereby introducing a single-stranded break in the target nucleic acid; contacting the target nucleic acid with a second effector protein described herein, to generate a second cleavage site in the target nucleic acid, ligating the regions flanking the first and second cleavage site, optionally through NHEJ or single- strand annealing, thereby resulting in the excision of a portion of the target nucleic acid between the first and second cleavage sites from the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally by HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., in between two cleavage sites). 130 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [510] In some embodiments, methods comprise editing a target nucleic acid with two or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof). In some embodiments, editing a target nucleic acid comprises introducing a two or more single-stranded breaks in a target nucleic acid. In some embodiments, a break is introduced by contacting a target nucleic acid with an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic acid binds to the effector protein and hybridizes to a region of the target nucleic acid, thereby recruits the effector protein to the region of the target nucleic acid. In some embodiments, binding of the effector protein to the guide nucleic acid and the region of the target nucleic acid activate the effector protein, and the activated effector protein introduces a break (e.g., a single stranded break) in the region of the target nucleic acid. In some embodiments, editing a target nucleic acid comprises introducing a first break in a first region of the target nucleic acid and a second break in a second region of the target nucleic acid. For example, in some embodiments, editing a target nucleic acid comprise contacting a target nucleic acid with a first guide nucleic acid that binds to a first effector protein and hybridizes to a first region of the target nucleic acid and a second guide nucleic acid that binds to a second effector protein or programmable nickase and hybridizes to a second region of the target nucleic acid. In some embodiments, the first effector protein introduces a first break in a first strand at the first region of the target nucleic acid, and the second effector protein introduces a second break in a second strand at the second region of the target nucleic acid. In some embodiments, a segment of the target nucleic acid between the first break and the second break is removed, thereby the target nucleic acid is edited. In some embodiments, a segment of the target nucleic acid between the first break and the second break is replaced (e.g., with donor nucleic acid), thereby the target nucleic acid is edited. [511] In some embodiments, methods, systems and compositions described herein edit a target nucleic acid wherein such editing results in one or more indels. In some embodiments, where compositions, systems, and/or methods described herein effect one or more indels, the impact on the transcription and/or translation of the target nucleic acid is predicted depending on: 1) the amount of indels generated; and 2) the location of the indel on the target nucleic acid. For example, as described herein, in some embodiments, if the amount of indels is not divisible by three, and the indels occur within or along a protein coding region, then the edit or mutation is a frameshift mutation. In some embodiments, if the amount of indels is divisible by three, then a frameshift mutation is not effected, but a splicing disruption mutation and/or sequence skip mutation is effected, such as an exon skip mutation. In some embodiments, if the amount of indels is not evenly divisible by three, then a frameshift mutation is effected. [512] In some embodiments, methods, systems and compositions described herein edit a target nucleic acid wherein such editing is measured by indel activity. Indel activity measures the amount of change in a target nucleic acid (e.g., nucleotide deletion(s) and/or insertion(s)) compared to a target nucleic acid that has not been contacted by a polypeptide described in compositions, systems, and methods described herein. For example, in some embodiments, indel activity is detected by next generation sequencing of one or more target loci of a target nucleic acid where indel percentage is calculated as the fraction of sequencing reads 131 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT containing insertions or deletions relative to an unedited reference sequence. In some embodiments, methods, systems, and compositions comprising polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) and guide nucleic acid described herein exhibit about 0.0001% to about 65% or more indel activity upon contact to a target nucleic acid compared to a target nucleic acid non-contacted with compositions, systems, or by methods described herein. For example, in some embodiments, methods, systems, and compositions comprising polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) and guide nucleic acid described herein exhibit about 0.0001%, about 0.001%, about 0.01%, about 0.1%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% or more indel activity. [513] In some embodiments, editing of a target nucleic acid as described herein effects one or more mutations comprising splicing disruption mutations, frameshift mutations (e.g., 1+ or 2+ frameshift mutation), sequence deletion, sequence skipping, sequence reframing, sequence knock-in, or any combination thereof. In some embodiments, the splicing disruption can be an editing that disrupts a splicing of a target nucleic acid or a splicing of a sequence that is transcribed from a target nucleic acid relative to a target nucleic acid without the splicing disruption. In some embodiments, the frameshift mutation can be an editing that alters the reading frame of a target nucleic acid relative to a target nucleic acid without the frameshift mutation. In some embodiments, the frameshift mutation can be a +2 frameshift mutation, wherein a reading frame is edited by 2 bases. In some embodiments, the frameshift mutation can be a +1 frameshift mutation, wherein a reading frame is edited by 1 base. In some embodiments, the frameshift mutation is an editing that alters the number of bases in a target nucleic acid so that it is not divisible by three. In some embodiments, the frameshift mutation can be an editing that is not a splicing disruption. In some embodiments a sequence as described in reference to the sequence deletion, sequence skipping, sequence reframing, and sequence knock-in can be a DNA sequence, a RNA sequence, an edited DNA or RNA sequence, a mutated sequence, a wild-type sequence, a coding sequence, a non-coding sequence, an exonic sequence (exon), an intronic sequence (intron), or any combination thereof. In some embodiments, the sequence deletion is an editing where one or more sequences in a target nucleic acid are deleted relative to a target nucleic acid without the sequence deletion. In some embodiments, the sequence deletion can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, the sequence deletion result in or effect a splicing disruption. In some embodiments, the sequence skipping is an editing where one or more sequences in a target nucleic acid are skipped upon transcription or translation of the target nucleic acid relative to a target nucleic acid without the sequence skipping. In some embodiments, the sequence skipping can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, the sequence skipping can result in or effect a splicing disruption. In some embodiments, the sequence reframing is an editing where one or more bases in a target are edited so that the reading frame of the sequence is reframed relative to a target nucleic acid without the sequence reframing. In some embodiments, the sequence reframing can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, the sequence reframing can result in or effect a frameshift mutation. In some 132 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT embodiments, the sequence knock-in is an editing where one or more sequences is inserted into a target nucleic acid relative to a target nucleic acid without the sequence knock-in. In some embodiments, the sequence knock-in can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, the sequence knock-in can result in or effect a splicing disruption. [514] In some embodiments, editing of a target nucleic acid can be locus specific, wherein compositions, systems, and methods described herein can edit a target nucleic acid at one or more specific loci to effect one or more specific mutations comprising splicing disruption mutations, frameshift mutations, sequence deletion, sequence skipping, sequence reframing, sequence knock-in, or any combination thereof. For example, editing of a specific locus can affect any one of a splicing disruption, frameshift (e.g., 1+ or 2+ frameshift), sequence deletion, sequence skipping, sequence reframing, sequence knock-in, or any combination thereof. In some embodiments, editing of a target nucleic acid can be locus specific, modification specific, or both. In some embodiments, editing of a target nucleic acid can be locus specific, modification specific, or both, wherein compositions, systems, and methods described herein comprise polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) described herein and a guide nucleic acid described herein. [515] In some embodiments, methods of editing a target nucleic acid or modulating the expression of a target nucleic acid is performed in vivo. In some embodiments, methods of editing a target nucleic acid or modulating the expression of a target nucleic acid is performed in vitro. For example, in some embodiments, a plasmid is edited in vitro using a composition described herein and introduced into a cell or organism. In some embodiments, methods of editing a target nucleic acid or modulating the expression of a target nucleic acid is performed ex vivo. For example, in some embodiments, methods comprise obtaining a cell from a subject, editing a target nucleic acid in the cell with methods described herein, and returning the cell to the subject. [516] In some embodiments, methods of modifying described herein comprise contacting a target nucleic acid with one or more components, compositions or systems described herein. In some embodiments, the one or more components, compositions or systems described herein comprise at least one of: a) one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof), or one or more nucleic acids encoding the one or more polypeptides; and b) one or more guide nucleic acids, or one or more nucleic acids encoding the one or more guide nucleic acids. In some embodiments, the one or more effector proteins introduce a single-stranded break or a double-stranded break in the target nucleic acid. In some embodiments, methods of modifying described herein produce a modified target nucleic acid comprising an engineered nucleic acid sequence that expresses polypeptide having new activity as compared to an unmodified target nucleic acid, or alters expression of an endogenous polypeptide as compared to an unmodified target nucleic acid. [517] In some embodiments, methods of modifying described herein comprise using one or more guide nucleic acids or uses thereof, wherein the methods modify a target nucleic acid at a single location. In some embodiments, the methods comprise contacting an RNP comprising polypeptides (e.g., effector proteins, 133 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT effector partners, fusion proteins, or combination thereof) and a guide nucleic acid to the target nucleic acid. In some embodiments, the methods introduce a mutation (e.g., point mutations, deletions) in the target nucleic acid relative to a corresponding wildtype nucleotide sequence. In some embodiments, the methods remove or correct a disease-causing mutation in a nucleic acid sequence to produce a corresponding wildtype nucleotide sequence. In some embodiments, the methods remove/correct point mutations, deletions, null mutations, or tissue-specific mutations in a target nucleic acid. In some embodiments, the methods introduce a single stranded cleavage, a nick, a deletion of one or two nucleotides, an insertion of one or two nucleotides, a substitution of one or two nucleotides, an epigenetic modification (e.g., methylation, demethylation, acetylation, or deacetylation), or a combination thereof to the target nucleic acid. In some embodiments, the methods comprise using an effector protein and two guide nucleic acids, wherein two RNPs cleave the target nucleic acid at the same location, wherein a first RNP comprises the effector protein and a first guide nucleic acid, and wherein a second RNP comprises the effector protein and a second guide nucleic acid. In some embodiments, methods comprising using two effector protein and two guide nucleic acids, wherein both RNPs cleave the target nucleic acid at the same location, wherein a first RNP comprises a first effector protein and a first target nucleic acid, and wherein a second RNP comprises a second effector protein and a second target nucleic acid. [518] In some embodiments, methods of modifying described herein comprise using one or more guide nucleic acids or uses thereof, wherein the methods modify a target nucleic acid at two different locations. In some embodiments, the methods introduce two cleavage sites in the target nucleic acid, wherein a first cleavage site and a second cleavage site comprise one or more nucleotides therebetween. In some embodiments, the methods cause deletion of the one or more nucleotides. In some embodiments, the deletion restores a wild-type reading frame. In some embodiments, the wild-type reading frame produces at least a partially functional protein. In some embodiments, the deletion causes a non-wild-type reading frame. In some embodiments, a non-wild-type reading frame produces a partially functional protein or non- functional protein. In some embodiments, the at least partially functional protein has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 180%, at least 200%, at least 300%, at least 400% activity compared to a corresponding wildtype protein. In some embodiments, the methods comprise using an effector protein and two guide nucleic acids, wherein two RNPs cleave the target nucleic acid at different locations, wherein a first RNP comprises the effector protein and a first guide nucleic acid, and wherein a second RNP comprises the effector protein and a second guide nucleic acid. In some embodiments, methods comprising using two effector protein and two guide nucleic acids, wherein both RNPs cleave the target nucleic acid at the same location, wherein a first RNP comprises a first effector protein and a first target nucleic acid, and wherein a second RNP comprises a second effector protein and a second target nucleic acid. [519] In some embodiments, methods of editing described herein comprise inserting a donor nucleic acid into a cleaved target nucleic acid. In some embodiments, the cleaved target nucleic acid formed by introducing a single-stranded break into a target nucleic acid. In some embodiments, the donor nucleic acid 134 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT is inserted at a specified (e.g., effector protein targeted) point within the target nucleic acid. In some embodiments, the cleaved target nucleic acid is cleaved at a single location. In such embodiments, the methods comprise contacting a target nucleic acid with an effector protein described herein, thereby introducing a single-stranded break in the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally by HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., at a cleavage site). In some embodiments, the cleaved target nucleic acid is cleaved at two locations. In such embodiments, the methods comprise contacting a target nucleic acid with an effector protein described herein, thereby introducing a single-stranded break in the target nucleic acid; contacting the target nucleic acid with a second effector protein described herein, to generate a second cleavage site in the target nucleic acid, ligating the regions flanking the first and second cleavage site, optionally through NHEJ or single-strand annealing, thereby resulting in the excision of a portion of the target nucleic acid between the first and second cleavage sites from the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally by HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., in between two cleavage sites). [520] In some embodiments, methods of modifying described herein comprise modifying a target nucleic acid in a cell. In some embodiments the methods of modifying described herein comprise contacting the cell with the any one of the compositions described herein, any one of the nucleic acid expression vectors described herein, any one of the pharmaceutical compositions described herein, or any one of the systems described herein, thereby modifying the target nucleic acid. In some embodiments, the methods of modifying described herein comprise cleaving at least one strand of a target nucleic acid, deleting or excising one or more nucleotides of the target nucleic acid, inserting one or more nucleotides into the target nucleic acid, substituting one or more nucleotides of the target nucleic acid with one or more alternative nucleotides, or combinations thereof. In some embodiments, the methods of modifying described herein comprise comprises deleting or excising one or more nucleotides of a target nucleic acid. In some embodiments, the methods of modifying described herein comprise the use of an additional engineered guide nucleic acid, or a nucleic acid encoding an additional engineered guide nucleic acid at least a portion of which hybridizes to a different target sequence of the target nucleic acid than the engineered guide nucleic acid. In some embodiments, the methods of modifying described herein comprise cleavage of two loci of a target nucleic acid and the one or more nucleotides between the two loci of the target nucleic acid are excised. In some embodiments, the one or more nucleotides to be deleted or excised is an expansion of a (CTG)
n repeat in the 3’ UTR of a human DMPK gene. In some embodiments, the expansion of the (CTG)
n is greater than about (CTG)
30. [521] In some embodiments, the methods of modifying described herein comprise modifying a target nucleic acid in a cell, wherein the cell is ex vivo or in vivo. In some embodiments, the cell is selected from an induced pluripotent stem cell (iPSC), a T cell, a hepatocyte, a cardiomyocyte, and a myoblast. Donor Nucleic Acids 135 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [522] In some embodiments, a donor nucleic acid comprises a nucleic acid that is incorporated into a target nucleic acid or genome. In some embodiments, a donor nucleic acid comprises a sequence that is derived from a plant, bacteria, fungi, virus, or an animal. In some embodiments, the animal is a non-human animal, such as, by way of non-limiting example, a mouse, rat, hamster, rabbit, pig, bovine, deer, sheep, goat, chicken, cat, dog, ferret, a bird, non-human primate (e.g., marmoset, rhesus monkey). In some embodiments, the non-human animal is a domesticated mammal or an agricultural mammal. In some embodiments, the animal is a human. In some embodiments, the sequence comprises a human wild-type (WT) gene or a portion thereof. In some embodiments, the human WT gene or the portion thereof comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% identical to an equal length portion of the WT sequence of any one of the sequences recited in TABLE 9. In some embodiments, the donor nucleic acid is incorporated into an insertion site of a target nucleic acid. [523] In some embodiments, the donor nucleic acid comprises single-stranded DNA or linear double- stranded DNA. In some embodiments, the donor nucleic acid comprises a nucleotide sequence encoding a functional polypeptide and/or wherein the donor nucleic acid comprises a wildtype sequence. In some embodiments, the donor nucleic acid comprises a protein coding sequence, a gene, a gene fragment, an exon, an intron, an exon fragment, an intron fragment, a gene regulatory fragment, a gene regulatory region fragment, coding sequences thereof, or combinations thereof. In some embodiments, the donor nucleic acid comprises a naturally occurring sequence. In some embodiments, the naturally occurring sequence does not contain a mutation. [524] In some embodiments, the donor nucleic acid comprises a gene fragment, an exon fragment, an intron fragment, a gene regulatory region fragment, or combinations thereof. In some embodiments, the fragment is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, or at least 80 contiguous nucleotides. [525] In some embodiments, a donor nucleic acid of any suitable size is integrated into a target nucleic acid or a genome. In some embodiments, the donor nucleic acid integrated into the target nucleic acid or the genome is less than 3, about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 kilobases in length. In some embodiments, the donor nucleic acid is more than 500 kilobases (kb) in length. [526] In some embodiments, a viral vector comprising a donor nucleic acid introduces the donor nucleic acid into a cell following transfection. In some embodiments, the donor nucleic acid is introduced into the cell by any mechanism of the transfecting viral vector, including, but not limited to, integration into the genome of the cell or introduction of an episomal plasmid or viral genome. [527] In some embodiments, an effector protein as described herein facilitates insertion of a donor nucleic acid at a site of cleavage or between two cleavage sites by cleaving (hydrolysis of a phosphodiester bond) of a nucleic acid resulting in a nick or double strand break – nuclease activity. 136 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [528] In some embodiments, a donor nucleic acid serves as a template in the process of homologous recombination, which carries an alteration that is to be or has been introduced into a target nucleic acid. By using the donor nucleic acid as a template, the genetic information, including the alteration, is copied into the target nucleic acid by way of homologous recombination. Genetically Modified Cells and Organisms [529] In some embodiments, methods of editing described herein is employed to generate a genetically modified cell. In some embodiments, the cell is a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., an archaeal cell). In some embodiments, the cell is derived from a multicellular organism and cultured as a unicellular entity. In some embodiments, the cell comprises a heritable genetic modification, such that progeny cells derived therefrom comprise the heritable genetic mutation. In some embodiments, the cell is progeny of a genetically modified cell comprising a genetic modification of the genetically modified parent cell. In some embodiments, the genetically modified cell comprises a deletion, insertion, mutation, or non-native sequence relative to a wild-type version of the cell or the organism from which the cell was derived. [530] In some embodiments, methods of editing described herein is performed in a cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is an isolated cell. In some embodiments, the cell is inside of an organism. In some embodiments, the cell is an organism. In some embodiments, the cell is in a cell culture. In some embodiments, the cell is one of a collection of cells. In some embodiments, the cell is a mammalian cell or derived there from. In some embodiments, the cell is a rodent cell or derived there from. In some embodiments, the cell is a human cell or derived there from. In some embodiments, the cell is a eukaryotic cell or derived there from. In some embodiments, the cell is a progenitor cell or derived there from. In some embodiments, the cell is a pluripotent stem cell or derived there from. In some embodiments, the cell is an animal cell or derived there from. In some embodiments, the cell is an invertebrate cell or derived there from. In some embodiments, the cell is a vertebrate cell or derived there from. In some embodiments, the cell is from a specific organ or tissue. In some embodiments, the cell is a hepatocyte. In some embodiments, the tissue is a subject’s blood, bone marrow, or cord blood. In some embodiments, the tissue is a heterologous donor blood, cord blood, or bone marrow. In some embodiments, the tissue is an allogenic blood, cord blood, or bone marrow. In some embodiments, the tissue comprises a muscle. In some embodiments, the muscle comprises a skeletal muscle. [531] In some embodiments, methods of editing described herein comprise contacting cells with compositions or systems described herein. In some embodiments, the contacting comprises electroporation, acoustic poration, optoporation, viral vector-based delivery, iTOP, nanoparticle delivery (e.g., lipid or gold nanoparticle delivery), cell-penetrating peptide (CPP) delivery, DNA nanostructure delivery, or any combination thereof. 137 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [532] In some embodiments, methods of editing described herein are performed in a subject. In some embodiments, the methods comprise administering compositions described herein to the subject. In some embodiments, the subject is a human. In some embodiments, the subject is a mammal (e.g., rat, mouse, cow, dog, pig, sheep, horse). In some embodiments, the subject is a vertebrate or an invertebrate. In some embodiments, the subject is a laboratory animal. In some embodiments, the subject is a patient. In some embodiments, the subject is at risk of developing, suffering from, or displaying symptoms of a disease. In some embodiments, the subject has a mutation associated with a gene described herein. In some embodiments, the subject displays symptoms associated with a mutation of a gene described herein. Methods of Detecting a Target Nucleic Acid [533] Provided herein are methods of detecting target nucleic acids. In some embodiments, methods comprise detecting target nucleic acids with compositions or systems described herein. In some embodiments, methods comprise detecting a target nucleic acid in a sample, e.g., a cell lysate, a biological fluid, or environmental sample. In some embodiments, methods comprise detecting a target nucleic acid in a cell. In some embodiments, methods of detecting a target nucleic acid in a sample or cell comprises contacting the sample or cell with an effector protein or a multimeric complex thereof, a guide nucleic acid, wherein at least a portion of the guide nucleic acid is complementary to at least a portion of the target nucleic acid, and a reporter nucleic acid that is cleaved in the presence of the effector protein, the guide nucleic acid, and the target nucleic acid, and detecting a signal produced by cleavage of the reporter nucleic acid, thereby detecting the target nucleic acid in the sample. In some embodiments, methods result in trans cleavage of the reporter nucleic acid. In some embodiments, methods result in cis cleavage of the reporter nucleic acid. In some embodiments, methods of detecting a target nucleic acid include a reporter nucleic acid comprising a detectable moiety that produces a detectable signal in the presence of the target nucleic acid, the effector protein, and the guide nucleic acid. [534] In some embodiments, the methods of detecting a target nucleic acid comprising: a) contacting the target nucleic acid with a composition comprising an effector protein as described herein, a guide nucleic acid as described herein, and a reporter nucleic acid that is cleaved in the presence of the effector protein, the guide nucleic acid, and the target nucleic acid; and b) detecting a signal produced by cleavage of the reporter nucleic acid, thereby detecting the target nucleic acid in the sample. In some embodiments, the methods result in trans cleavage of the reporter nucleic acid. In some embodiments, the methods result in cis cleavage of the reporter nucleic acid. In some embodiments, the reporter nucleic acid is a single stranded nucleic acid. In some embodiments, the reporter comprises a detection moiety. In some embodiments, the reporter nucleic acid is capable of being cleaved by the effector protein. In some embodiments, a cleaved reporter nucleic acid generates a detectable product or a first detectable signal. In some embodiments, the first detectable signal is a change in color. In some embodiments, the change is color is measured indicating presence of the target nucleic acid. In some embodiments, the first detectable signal is measured on a support medium. 138 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [535] In some embodiments, methods of detecting comprise contacting a target nucleic acid, a cell comprising the target nucleic acid, or a sample comprising a target nucleic acid with an effector protein that comprises an amino acid sequence that is at least is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences recited in TABLE 1 or a variant thereof. In some embodiments, the amino acid sequence of the effector protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences recited in TABLE 1 or a variant thereof. In some embodiments, the effector protein comprising an amino acid sequence that is at least 90% identical to a sequence selected from any one of the sequences recited in TABLE 1 or a variant thereof. [536] In some embodiments, methods comprise contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and an effector protein that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some protein-nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. [537] In some embodiments, methods comprise contacting the sample comprising the target nucleic acid with a guide nucleic acid targeting a target nucleic acid segment, an effector protein capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment, a single stranded nucleic acid of a reporter comprising a detection moiety, wherein the nucleic acid of a reporter is capable of being cleaved by the activated effector protein, thereby generating a first detectable signal, cleaving the single stranded nucleic acid of a reporter using the effector protein that cleaves as measured by a change in color, and measuring the first detectable signal on the support medium. [538] In some embodiments, methods comprise contacting the sample or cell with an effector protein or a multimeric complex thereof and a guide nucleic acid at a temperature of at least about 25°C, at least about 30°C, at least about 35°C, at least about 37°C, at least about 40°C, at least about 50°C, at least about 65°C, at least about 70°C, or at least about 75°C. In some embodiments, the temperature is not greater than 80°C. In some embodiments, the temperature is about 25°C, about 30°C, about 35°C, at least about 37°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, or about 90°C. In some embodiments, the temperature is about 25°C to about 45°C, about 35°C to about 55°C, about 37°C to about 60°C, or about 55°C to about 65°C. In some embodiments, the temperature is about 37°C to about 45°C, about 37°C to about 50°C, about 37°C to about 55°C, about 37°C to about 60°C, or about 37°C to about 65°C. [539] In some embodiments, methods comprise contacting the sample or cell with an effector protein or a multimeric complex thereof and a guide nucleic acid in the presence of salts (e.g., compositions comprising salts). In some embodiments, the method comprises a solution, wherein the solution comprises 139 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT one or more salt. Accordingly, in some embodiments, the salt comprises one or more salt selected from a magnesium salt, a zinc salt, a potassium salt, a calcium salt, and a sodium salt. In some embodiments, the salt is a combination of two or more salts. For example, in some embodiments, the salt is a combination of two or more salts selected from a magnesium salt, a zinc salt, a potassium salt, a calcium salt and a sodium salt. In some embodiments, the salt is magnesium acetate. In some embodiments, the salt is magnesium chloride. In some embodiments, the salt is potassium acetate. In some embodiments, the salt is potassium nitrate. In some embodiments, the salt is zinc chloride. In embodiments, the salt is sodium chloride. In some embodiments, the salt is potassium chloride. In some embodiments, the concentration of the one or more salt in the solution is about 0.001 mM to about 500 mM. In some embodiments, the concentration of the salt is about 0.001 mM to about 400 mM. In some embodiments, the concentration of the salt is about 0.001 mM to about 300 mM. In some embodiments, the concentration of the salt is about 0.001 mM to about 200 mM. In some embodiments, the concentration of the salt is about 0.001 mM to about 100 mM. In some embodiments, the concentration of the salt is about 0.001 mM to about 10 mM. In some embodiments, the concentration of the salt is about 0.01 mM to about 500 mM. In some embodiments, the concentration of the salt is about 0.01 mM to about 400 mM. In some embodiments, the concentration of the salt is about 0.01 mM to about 300 mM. In some embodiments, the concentration of the salt is about 0.01 mM to about 200 mM. In some embodiments, the concentration of the salt is about 0.01 mM to about 100 mM. In some embodiments, the concentration of the salt is about 0.01 mM to about 10 mM. In some embodiments, the concentration of the salt is about 0.1 mM to about 500 mM. In some embodiments, the concentration of the salt is about 0.1 mM to about 400 mM. In some embodiments, the concentration of the salt is about 0.1 mM to about 300 mM. In some embodiments, the concentration of the salt is about 0.1 mM to about 200 mM. In some embodiments, the concentration of the salt is about 0.1 mM to about 100 mM. In some embodiments, the concentration of the salt is about 0.1 mM to about 10 mM. In some embodiments, the concentration of the salt is about 1 mM to about 500 mM. In some embodiments, the concentration of the salt is about 1 mM to about 400 mM. In some embodiments, the concentration of the salt is about 1 mM to about 300 mM. In some embodiments, the concentration of the salt is about 1 mM to about 200 mM. In some embodiments, the concentration of the salt is about 1 mM to about 100 mM. In some embodiments, the concentration of the salt is about 1 mM to about 10 mM. In some embodiments, the concentration of the salt is about 10 mM to about 500 mM. In some embodiments, the concentration of the salt is about 10 mM to about 400 mM. In some embodiments, the concentration of the salt is about 10 mM to about 300 mM. In some embodiments, the concentration of the salt is about 10 mM to about 200 mM. In some embodiments, the concentration of the salt is about 10 mM to about 100 mM. In some embodiments, the concentration of the salt is about 100 mM to about 500 mM. In some embodiments, the concentration of the salt is about 100 mM to about 400 mM. In some embodiments, the concentration of the salt is about 100 mM to about 300 mM. In some embodiments, the concentration of the salt is about 100 mM to about 200 mM. In some embodiments, the salt is potassium acetate and the concentration of salt in the solution is about 100 mM. In some embodiments, the salt is potassium acetate or sodium chloride 140 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT and the concentration of salt in the solution is about 200 mM. In some embodiments, the salt is potassium acetate or sodium chloride and the salt of potassium in the solution is about 100 mM to about 200 mM. [540] In some embodiments, methods of detecting a target nucleic acid by a cleavage assay. In some embodiments, the target nucleic acid is a single-stranded target nucleic acid. In some embodiments, the cleavage assay comprises: a) contacting the target nucleic acid with a composition comprising an effector protein as described; and b) cleaving the target nucleic acid. In some embodiments, the cleavage assay comprises an assay designed to visualize, quantitate or identify cleavage of a nucleic acid. In some embodiments, the method is an in vitro trans cleavage assay. In some embodiments, a cleavage activity is a trans cleavage activity. In some embodiments, the method is an in vitro cis cleavage assay. In some embodiments, a cleavage activity is a cis cleavage activity. In some embodiments, the cleavage assay follows a procedure comprising: (i) providing a composition comprising an equimolar amounts of an effector protein as described herein, and a guide nucleic acid described herein, under conditions to form an RNP complex; (ii) adding a plasmid comprising a target nucleic acid, wherein the target nucleic acid is a linear dsDNA, wherein the target nucleic acid comprises a target sequence and a PAM (iii) incubating the mixture under conditions to enable cleavage of the plasmid; (iv) quenching the reaction with EDTA and a protease; and (v) analyzing the reaction products (e.g., viewing the cleaved and uncleaved linear dsDNA with gel electrophoresis). [541] In some embodiments, there is a threshold of detection for methods of detecting target nucleic acids. In some embodiments, methods are not capable of detecting target nucleic acids that are present in a sample or solution at a concentration less than or equal to 10 nM. For example, when a threshold of detection is 10 nM, then a signal can be detected when a target nucleic acid is present in the sample at a concentration of 10 nM or more. In some embodiments, the threshold of detection is less than or equal to 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.005 nM, 0.001 nM, 0.0005 nM, 0.0001 nM, 0.00005 nM, 0.00001 nM, 10 pM, 1 pM, 500 fM, 250 fM, 100 fM, 50 fM, 10 fM, 5 fM, 1 fM, 500 attomole (aM), 100 aM, 50 aM, 10 aM, or 1 aM. In some embodiments, the threshold of detection is in a range of from 1 aM to 1 nM, 1 aM to 500 pM, 1 aM to 200 pM, 1 aM to 100 pM, 1 aM to 10 pM, 1 aM to 1 pM, 1 aM to 500 fM, 1 aM to 100 fM, 1 aM to 1 fM, 1 aM to 500 aM, 1 aM to 100 aM, 1 aM to 50 aM, 1 aM to 10 aM, 10 aM to 1 nM, 10 aM to 500 pM, 10 aM to 200 pM, 10 aM to 100 pM, 10 aM to 10 pM, 10 aM to 1 pM, 10 aM to 500 fM, 10 aM to 100 fM, 10 aM to 1 fM, 10 aM to 500 aM, 10 aM to 100 aM, 10 aM to 50 aM, 100 aM to 1 nM, 100 aM to 500 pM, 100 pM to 200 pM, 100 aM to 100 pM, 100 aM to 10 pM, 100 aM to 1 pM, 100 aM to 500 fM, 100 aM to 100 fM, 100 aM to 1 fM, 100 aM to 500 aM, 500 aM to 1 nM, 500 aM to 500 pM, 500 aM to 200 pM, 500 aM to 100 pM, 500 aM to 10 pM, 500 aM to 1 pM, 500 aM to 500 fM, 500 aM to 100 fM, 500 aM to 1 fM, 1 fM to 1 nM, 1 fM to 500 pM, 1 fM to 200 pM, 1 fM to 100 pM, 1 fM to 10 pM, 1 fM to 1 pM, 10 fM to 1 nM, 10 fM to 500 pM, 10 fM to 200 pM, 10 fM to 100 pM, 10 fM to 10 pM, 10 fM to 1 pM, 500 fM to 1 nM, 500 fM to 500 pM, 500 fM to 200 pM, 500 fM to 100 pM, 500 fM to 10 pM, 500 fM to 1 pM, 800 fM to 1 nM, 800 fM to 500 pM, 800 fM to 200 pM, 800 fM to 100 pM, 800 fM to 10 pM, 800 fM to 1 pM, 1 pM to 1 nM, 1 pM to 500 pM, 1 pM to 200 pM, 1 pM to 100 pM, or 141 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT 1 pM to 10 pM. In some embodiments, the threshold of detection in a range of from 800 fM to 100 pM, 1 pM to 10 pM, 10 fM to 500 fM, 10 fM to 50 fM, 50 fM to 100 fM, 100 fM to 250 fM, or 250 fM to 500 fM. In some embodiments, the threshold of detection is in a range of from 2 aM to 100 pM, from 20 aM to 50 pM, from 50 aM to 20 pM, from 200 aM to 5 pM, or from 500 aM to 2 pM. [542] In some embodiments, the target nucleic acid is present in a cleavage reaction at a concentration of about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 µM, about 10 µM, or about 100 µM. In some embodiments, the target nucleic acid is present in a cleavage reaction at a concentration of from 10 nM to 20 nM, from 20 nM to 30 nM, from 30 nM to 40 nM, from 40 nM to 50 nM, from 50 nM to 60 nM, from 60 nM to 70 nM, from 70 nM to 80 nM, from 80 nM to 90 nM, from 90 nM to 100 nM, from 100 nM to 200 nM, from 200 nM to 300 nM, from 300 nM to 400 nM, from 400 nM to 500 nM, from 500 nM to 600 nM, from 600 nM to 700 nM, from 700 nM to 800 nM, from 800 nM to 900 nM, from 900 nM to 1 µM, from 1 µM to 10 µM, from 10 µM to 100 µM, from 10 nM to 100 nM, from 10 nM to 1 µM, from 10 nM to 10 µM, from 10 nM to 100 µM, from 100 nM to 1 µM, from 100 nM to 10 µM, from 100 nM to 100 µM, or from 1 µM to 100 µM. In some embodiments, the target nucleic acid is present in a cleavage reaction at a concentration of from 20 nM to 50 µM, from 50 nM to 20 µM, or from 200 nM to 5 µM. [543] In some embodiments, methods detect a target nucleic acid in less than 60 minutes. In some embodiments, methods detect a target nucleic acid in less than about 120 minutes, less than about 110 minutes, less than about 100 minutes, less than about 90 minutes, less than about 80 minutes, less than about 70 minutes, less than about 60 minutes, less than about 55 minutes, less than about 50 minutes, less than about 45 minutes, less than about 40 minutes, less than about 35 minutes, less than about 30 minutes, less than about 25 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, or less than about 1 minute. [544] In some embodiments, methods require at least about 120 minutes, at least about 110 minutes, at least about 100 minutes, at least about 90 minutes, at least about 80 minutes, at least about 70 minutes, at least about 60 minutes, at least about 55 minutes, at least about 50 minutes, at least about 45 minutes, at least about 40 minutes, at least about 35 minutes, at least about 30 minutes, at least about 25 minutes, at least about 20 minutes, at least about 15 minutes, at least about 10 minutes, or at least about 5 minutes to detect a target nucleic acid. In some embodiments, the sample is contacted with the reagents for from 5 minutes to 120 minutes, from 5 minutes to 100 minutes, from 10 minutes to 90 minutes, from 15 minutes to 45 minutes, or from 20 minutes to 35 minutes. [545] In some embodiments, methods of detecting are performed in less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 50 minutes, less than 45 minutes, less than 40 minutes, less than 35 minutes, less than 30 minutes, less than 25 minutes, less than 20 minutes, less than 15 minutes, less 142 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, or less than 5 minutes. In some embodiments, methods of detecting are performed in about 5 minutes to about 10 hours, about 10 minutes to about 8 hours, about 15 minutes to about 6 hours, about 20 minutes to about 5 hours, about 30 minutes to about 2 hours, or about 45 minutes to about 1 hour. [546] In some embodiments, methods comprise detecting a detectable signal within 5 minutes of contacting the sample and/or the target nucleic acid with the guide nucleic acid and/or the effector protein. In some embodiments, detecting occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, or 120 minutes of contacting the target nucleic acid. In some embodiments, detecting occurs within 1 to 120, 5 to 100, 10 to 90, 15 to 80, 20 to 60, or 30 to 45 minutes of contacting the target nucleic acid. [547] In some embodiments, methods of detecting as disclosed herein are compatible with methods for diagnosis of a disease or disorder. Amplification of a Target Nucleic Acid [548] Methods comprise amplifying a target nucleic acid for detection using any of the compositions or systems described herein. In some embodiments, amplifying comprises changing the temperature of the amplification reaction, also known as thermal amplification (e.g., PCR). In some embodiments, amplifying is performed at essentially one temperature, also known as isothermal amplification. In some embodiments, amplifying improves at least one of sensitivity, specificity, or accuracy of the detection of the target nucleic acid. [549] In some embodiments, amplifying comprises subjecting a target nucleic acid to an amplification reaction selected from transcription mediated amplification (TMA), helicase dependent amplification (HDA), or circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), and improved multiple displacement amplification (IMDA), or any one of the amplification methods described herein. [550] In some embodiments, amplification of the target nucleic acid comprises modifying the sequence of the target nucleic acid. For example, in some embodiments, amplification is used to insert a PAM sequence into a target nucleic acid that lacks a PAM sequence. In some embodiments, amplification is used to increase the homogeneity of a target nucleic acid in a sample. For example, in some embodiments, amplification is used to remove a nucleic acid variation that is not of interest in the target nucleic acid. 143 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [551] In some embodiments, amplifying takes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes. In some embodiments, amplifying is performed at a temperature of around 20-45ºC. In some embodiments, amplifying is performed at a temperature of less than about 20ºC, less than about 25ºC, less than about 30ºC, less than about 35ºC, less than about 37ºC, less than about 40ºC, or less than about 45ºC. In some embodiments, the nucleic acid amplification reaction is performed at a temperature of at least about 20ºC, at least about 25ºC, at least about 30ºC, at least about 35ºC, at least about 37ºC, at least about 40ºC, or at least about 45ºC. Detection of a Target Nucleic Acid [552] Described herein are various methods of sample amplification and detection in a single reaction volume. In some embodiments, methods include simultaneous amplification and detection in the same volume and/or in the same reaction. In some embodiments, methods include sequential amplification and detection in the same volume. In some embodiments, amplification and detection occur in a single reaction, where reverse transcription, amplification, in vitro transcription, or any combination thereof, and detection are carried out in a single volume. Any suitable method of reverse transcription, amplification, in vitro transcription, and detection can be used in such a reaction, such as methods of reverse transcription, amplification, in vitro transcription, and detection described herein. [553] In some embodiments, a DETECTR reaction is used for detecting the presence of a specific target gene in the same. In some embodiments, the DETECTR reaction produces a detectable signal, as described elsewhere herein, in the presence of a target nucleic acid sequence comprising a target gene. In some embodiments, the DETECTR reaction does not produce a signal in the absence of the target nucleic acid or in the presence of a nucleic acid sequence that does not comprise the specific mutation or comprises a different mutation. In some embodiments the mutation is a SNP. In some embodiments, a DETECTR reaction comprises a guide RNA reverse complementary to a portion of a target nucleic acid sequence comprising a specific SNP. In some embodiments, the guide RNA and the target nucleic acid comprising the specific SNP bind to and activate an effector protein, thereby producing a detectable signal as described elsewhere herein. In some embodiments, the guide RNA and a nucleic acid sequence that does not comprise the specific SNP does not bind to or activate the effector protein and does not produce a detectable signal. In some embodiments, a target nucleic acid sequence, that may or may not comprise a specific SNP, is amplified using any amplification method disclosed herein. In some embodiments, the amplification reaction is combined with a reverse transcription reaction, a DETECTR reaction, or both. In some embodiments, the target nucleic acid sequence can comprise a SNP. In some embodiments, the target nucleic acid sequence can comprise a sequence indicative of a human disease. [554] In some embodiments, a DETECTR reaction, as described elsewhere herein, produces a detectable signal specifically in the presence of a target nucleic acid sequence comprising a target gene. In some embodiments, the target nucleic acid sequence can comprise a sequence indicative of a human disease. In some embodiments, the detectable signal produced in the DETECTR reaction is higher in the presence of 144 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT a target nucleic acid comprising target nucleic acid than in the presence of a nucleic acid that does not comprise the target nucleic acid. In some embodiments, the DETECTR reaction produces a detectable signal that is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at last 400-fold, at least 500-fold, at least 1000-fold, at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000-fold, at least 9000-fold, at least 10000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold greater in the presence of a target nucleic acid comprising a target nucleic acid than in the presence of a nucleic acid that does not comprise the target nucleic acid. In some embodiments, the DETECTR reaction produces a detectable signal that is from 1-fold to 2-fold, from 2-fold to 3-fold, from 3-fold to 4-fold, from 4-fold to 5-fold, from 5-fold to 10-fold, from 10-fold to 20-fold, from 20-fold to 30-fold, from 30-fold to 40-fold, from 40-fold to 50-fold, from 50-fold to 100-fold, from 100-fold to 500-fold, from 500-fold to 1000-fold, from 1000-fold to 10,000-fold, from 10,000-fold to 100,000-fold, or from 100,000-fold to 1,000,000-fold greater in the presence of a target nucleic acid comprising a specific mutation or SNP than in the presence of a nucleic acid that does not comprise the specific mutation or SNP. In some embodiments, the target nucleic acid sequence can comprise a SNP. In some embodiments, the target nucleic acid sequence can comprise a sequence indicative of a human disease. [555] In some embodiments, a DETECTR reaction is used for detecting the presence of a target nucleic acid associated with a disease or a condition in a nucleic acid sample. In some embodiments, the DETECTR reaction reaches signal saturation within about 30 seconds, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 75 minutes, about 80 minutes, or about 85 minutes and be used to detect the presence of a target gene associated with an increased likelihood of developing a disease or a condition in a nucleic acid sample. In some embodiments, the DETECTR reaction is used for detecting the presence of a target gene associated with a phenotype in a nucleic acid sample. For example, in some embodiments, a DETECTR reaction is used for detecting a target nucleic acid (e.g., gene or exon), or a mutation of a target nucleic acid(e.g., a SNP), as recited in TABLE 9, 9.1, or 9.2. In another example, in some embodiments, a DETECTR reaction is used for detecting a target nucleic acid or a mutation of a target nucleic acid associated with any one of the diseases or disorders recited in TABLE 10. In some embodiments, a DETECTR reaction is used for detecting a SNP associated with a phenotype, for example, a phenotype associated with DMPK. Methods of Treating a Disease or Disorder [556] Described herein are methods for treating a disease in a subject by contacting a target nucleic acid with a composition or system described herein, wherein the target nucleic acid is associated with a gene or expression of a gene related to the disease. In some embodiments, methods comprise treating, preventing, or inhibiting a disease or disorder associated with a mutation or aberrant expression of a gene. In some 145 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT embodiments, methods for treating a disease or disorder comprise methods of editing a nucleic acid described herein. [557] In some embodiments, methods comprise administration of a composition(s) or component(s) of a system described herein. In some embodiments, the composition(s) or component(s) of the system comprises use of a recombinant nucleic acid (DNA or RNA), administered for the purpose to edit a nucleic acid. In some embodiments, the composition or component of the system comprises use of a vector to introduce a functional gene or transgene. In some embodiments, vectors comprise nonviral vectors, including cationic polymers, cationic lipids, or bio-responsive polymers. In some embodiments, the bio- responsive polymer exploits chemical-physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space. In some embodiments, vectors comprise viral vectors, including retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses. In some embodiments, the vector comprises a replication-defective viral vector, comprising an insertion of a therapeutic gene inserted in genes essential to the lytic cycle, preventing the virus from replicating and exerting cytotoxic effects. By way of non-limiting example, in some embodiments, the composition(s) comprises pharmaceutical compositions described herein. Methods of gene therapy that are applicable to the compositions and systems described herein are described in more detail in Ingusci et al., “Gene Therapy Tools for Brain Diseases”, Front. Pharmacol.10:724 (2019), which is hereby incorporated by reference in its entirety. [558] In some embodiments, treating, preventing, or inhibiting disease or disorder in a subject comprises contacting a target nucleic acid associated with a particular ailment with a composition described herein. In some embodiments, the methods of treating, preventing, or inhibiting a disease or disorder involves removing, editing, modifying, replacing, transposing, or affecting the regulation of a genomic sequence of a patient in need thereof. In some embodiments, the methods of treating, preventing, or inhibiting a disease or disorder involves modulating gene expression. [559] In some embodiments, the compositions and systems described herein are for use in therapy. In some embodiments, the compositions and systems described herein are for use in treating a disease or condition described herein. Also provided is the use of the compositions described herein in the manufacture of a medicament. Also provided is the use of the compositions described herein in the manufacture of a medicament for therapeutic and/or prophylactic treatment of a disease or condition described herein. [560] In some embodiments, the polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) described herein are for use in therapy. In some embodiments, the polypeptides described herein are for use in treating a disease or condition described herein. Also provided is the use of the polypeptides described herein in the manufacture of a medicament. Also provided is the use of the polypeptides described herein in the manufacture of a medicament for therapeutic and/or prophylactic treatment of a disease or condition described herein. 146 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [561] In some embodiments, the guide nucleic acids described herein are for use in therapy. In some embodiments, the guide nucleic acids described herein are for use in treating a disease or condition described herein. Also provided is the use of the guide nucleic acids described herein in the manufacture of a medicament. Also provided is the use of the guide nucleic acids described herein in the manufacture of a medicament for therapeutic and/or prophylactic treatment of a disease or condition described herein. [562] Described herein are compositions, systems, and methods for treating a disease in a subject by editing a target nucleic acid associated with a gene or expression of a gene related to the disease. For example, in some embodiments, the editing comprises knock-out of a gene comprising the target nucleic acid. In some embodiments, the compositions, systems and methods comprise LNPs, wherein the LNPs comprise the effector proteins described herein or nucleic acids encoding the effector proteins, the effector partners described herein or nucleic acids encoding the effector partners, the fusion proteins described herein or nucleic acids encoding the fusion proteins, or combinations thereof. In some embodiments, the LNPs comprise chemically modified guide nucleic acids. In some embodiments, the LNPs described herein are used for delivering the compositions, or one or more components of the systems described herein to a specific organ (e.g., liver). Alternatively, in some embodiments, the compositions, systems and methods comprise AAV particles, wherein the AAV particles comprise nucleic acids encoding the effector proteins described herein, the effector partners described herein, the fusion proteins described herein, or combinations thereof. In some embodiments, the AAV particles comprise nucleic acids encoding guide nucleic acids described herein. In some embodiments, the AAV particles described herein are used for delivering the compositions, or one or more components of the systems described herein to a specific cells (e.g., nerve cells or muscle cells). In some embodiments, methods comprise administering a composition or cell described herein to a subject. By way of non-limiting example, in some embodiments, the disease comprises a cancer, an ophthalmological disorder, a neurological disorder, a neurodegenerative disease, a blood disorder, or a metabolic disorder, or a combination thereof. In some embodiments, the disease comprises an inherited disorder, also referred to as a genetic disorder. In some embodiments, the disease is the result of an infection or associated with an infection. Also, by way of non-limiting example, the compositions are pharmaceutical compositions described herein. [563] In some embodiments, the compositions and methods described herein are used for treating, preventing, or inhibiting a disease or syndrome in a subject. In some embodiments, the disease is a liver disease, a lung disease, an eye disease, or a muscle disease. Exemplary diseases and syndromes include but are not limited to the diseases and syndromes listed in TABLE 10. [564] Described herein are methods for treating a disease in a subject, wherein the method comprises administering the compositions described herein to the subject. In some embodiments, the compositions described herein modify or edit at least one target nucleic acid associated with a disease described herein or the expression thereof. In some embodiments, the target nucleic acid is any one of the target nucleic acids listed in TABLE 9 or 9.1. In some embodiments, the target nucleic acid is a human DMPK gene. In some embodiments, the target nucleic acid comprises a mutation relative to the wild-type DMPK gene. In 147 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT some embodiments, the compositions modify or edit one or more nucleotides of a target nucleic acid. In some embodiments, modifying comprises cleaving at least one strand of a target nucleic acid, deleting or excising one or more nucleotides of the target nucleic acid, inserting one or more nucleotides into the target nucleic acid, substituting one or more nucleotides of the target nucleic acid with one or more alternative nucleotides, or combinations thereof. In some embodiments, the compositions cleave two loci of a target nucleic acid, and wherein the composition excises one or more nucleotides between the two loci of the target nucleic acid. In some embodiments, modifying comprises deleting or excising one or more nucleotides of a target nucleic acid, wherein the one or more nucleotides are located in an untranslated region, protein coding region, an exon, an intron, a gene regulatory region, coding sequences thereof, or combinations thereof. In some embodiments, the one or more nucleotides are located in any one of the locations listed in TABLE 9.2. In some embodiments, the compositions are the pharmaceutical compositions described herein. In some embodiments, the disease is any one of the diseases recited in TABLE 10. In some embodiments, the disease is DM1. In some embodiments, the disease is associated with an expansion of a (CTG)
n repeat in the 3'-untranslated region (UTR) of a human DMPK gene. In some embodiments, the expansion of the (CTG)
n repeat in the 3'-untranslated region (UTR) of a human DMPK gene are greater than about (CTG)
30. In some embodiments, the repeats of (CTG)
n are greater than about (CTG)
40. In some embodiments, the repeats of (CTG)
n are greater than about (CTG)
50. In some embodiments, the repeats of (CTG)
n are about (CTG)
50 to about (CTG)
5,000. In some embodiments, the expansion of the (CTG)
n repeat is associated with a frame shift reading mutation. In some embodiments, the expansion of the (CTG)
n repeat is associated with exponential upregulation of DMPK transcription. In some embodiments, the expansion of the (CTG)
n repeat is associated with exponential increase of DMPK RNA transcript (e.g., mRNA), which can result in the RNA-associated toxicity, causing the DM1 disease. Disease, Cell, or Tissue Specific System [565] Described herein are methods for treating, preventing, or inhibiting a disease or disorder in a subject by contacting a target nucleic acid associated with a disease, cell, or tissue-specific system described herein, wherein the target nucleic acid is associated with a gene or expression of a gene related to the disease or disorder. In some embodiments, methods comprise treating, preventing, or inhibiting a disease or disorder associated with a mutation or aberrant expression of a gene. Methods can comprise administration of a composition(s) or component(s) of a system described herein. By way of non-limiting example, the composition(s) comprise pharmaceutical compositions described herein. Methods for treating a disease or disorder can also involve contacting a cell comprising a target nucleic acid with a disease, cell, or tissue- specific system as described herein. Methods for treating a disease in a subject described herein can also comprise administration of a composition(s) or component(s) of a disease, cell, or tissue-specific system further comprising a vector system or a vector delivery system, as described herein. Methods for treating a disease in a subject described herein can also comprise administration of a composition(s) or component(s) of a disease, cell, or tissue-specific system such as a gene therapy system as described herein. Disease, Cell, or Tissue Specific Vector 148 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [566] Compositions, systems, and methods described herein can comprise a disease, cell, or tissue- specific vector or a use thereof, as described herein. A disease, cell, or tissue-specific vector can comprise a nucleic acid of interest. In some embodiments, the nucleic acid of interest comprises one or more components of a composition or system described herein. In some embodiments, the nucleic acid of interest comprises a nucleotide sequence that encodes one or more components of the composition or system described herein. In some embodiments, one or more components comprises a polypeptide(s), guide nucleic acid(s), target nucleic acid(s), and donor nucleic acid(s). In some embodiments, the component comprises a nucleic acid encoding an effector protein, a donor nucleic acid, and a guide nucleic acid or a nucleic acid encoding the guide nucleic acid. In some embodiments, a disease, cell, or tissue-specific vector is part of a vector or delivery vector system. The vector system can comprise a library of vectors each encoding one or more component of a composition or system described herein. In some embodiments, components described herein (e.g., an effector protein, a guide nucleic acid, and/or a target nucleic acid) are encoded by the same disease, cell, or tissue-specific vector. In some embodiments, components described herein (e.g., an effector protein, a guide nucleic acid, and/or a target nucleic acid) are each encoded by different disease, cell, or tissue-specific vectors of the system. In some embodiments, a disease, cell, or tissue-specific vector encoding a donor nucleic acid further encodes a target nucleic acid. [567] In some embodiments, a disease, cell, or tissue-specific vector comprises a nucleotide sequence encoding one or more effector proteins as described herein. In some embodiments, the one or more effector proteins comprise at least two effector proteins. In some embodiments, the at least two effector protein are the same. In some embodiments, the at least two effector proteins are different from each other. In some embodiments, the nucleotide sequence is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell. In some embodiments, the disease, cell, or tissue-specific vector comprises the nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more effector proteins. [568] In some embodiments, a disease, cell, or tissue-specific vector encodes one or more of any system components, including but not limited to effector proteins, guide nucleic acids, donor nucleic acids, and target nucleic acids as described herein. In some embodiments, a system component encoding sequence is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell. In some embodiments, a disease, cell, or tissue-specific vector encodes 1, 2, 3, 4 or more of any system components. For example, a disease, cell, or tissue-specific vector can encode two or more guide nucleic acids, wherein each guide nucleic acid comprises a different sequence. A disease, cell, or tissue-specific vector can encode an effector protein and a guide nucleic acid. A disease, cell, or tissue-specific vector can encode an effector protein, a guide nucleic acid, a donor nucleic acid, or combinations thereof. In some embodiments, a disease, cell, or tissue-specific vector comprises any of the components that can be comprised in any of the vectors described herein. In some embodiments, a disease, cell, or tissue-specific vector is any of the vectors described herein (e.g., viral, non-viral). 149 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT Gene Therapy System [569] In some embodiments, a method of treating a disease or disorder in a subject described herein comprises administration of a composition(s) comprising a gene therapy system desired herein or component(s) of a gene therapy system as described herein. Gene therapy systems encompassed by the present disclosure are also known as virus-directed enzyme prodrug therapy, suicide gene therapy, and gene prodrug activation therapy. A gene therapy system described herein can comprise a nucleic acid encoding an effector protein or enzyme with disease, cell, or tissue-specific expression as described herein. In some embodiments, a gene therapy system described herein comprises a nucleic acid expression vector as described herein comprising the nucleic acid encoding for an effector protein sequence, and a promoter, optionally wherein the promoter comprises cell-specific or restricted expression. In some embodiments, gene therapy systems described herein are delivered to a cell, optionally delivered as a composition to a cell, in a manner suitable to direct the gene therapy system into a cell, which can be a diseased cell, tumor cell, cancer cell, or combinations thereof. In some embodiments, promoter expression is induced such that the effector protein is expressed inside the cell. In some embodiments, the cell is a tumor cell wherein the tumor cell environment activates the expression of the vector’s promoter sequence and thus the intracellular expression of the effector protein. In some embodiments, activity by the expressed effector protein or enzyme within the cell induces cell signaling cascades and/or cell death as described herein. Cancer [570] In some embodiments, the disease comprises cancer. Non-limiting examples of cancers include: adrenocortical carcinoma; basal-cell carcinoma; extrahepatic (cholangiocarcinoma); breast cancer; carcinoma of adult, unknown primary site; carcinoma of unknown primary; childhood adrenocortical carcinoma; colorectal cancer; hepatocellular (liver cancer); islet cell carcinoma (endocrine pancreas); liver cancer (primary); lung cancer, non-small cell; lung cancer, small cell; lymphoma, primary central nervous system; male breast cancer; Merkel cell cancer; Merkel cell skin carcinoma; nasopharyngeal carcinoma; neuroblastoma, non-small cell lung cancer; renal cell carcinoma (kidney cancer); NUT midline carcinoma; small cell lung cancer; squamous cell carcinoma; thymoma and thymic carcinoma; adenoid cystic carcinoma; bronchiolo-alveolar adenocarcinoma; basal cell carcinoma; basal cell carcinoma 1; colon adenocarcinoma; breast adenocarcinoma; gastric cancer; gastric adenocarcinoma; germ cell cancer; muscle cancer; ovarian cancer; ovarian serous cystadenocarcinoma; pancreatic cancer; prostate cancer; skin carcinoma; testicular germ cell cancer; and thyroid cancer. Illustrative Embodiments [571] The present disclosure provides the following illustrative embodiments. [572] Embodiment 1. A composition comprising: (a) a polypeptide or a nucleic acid encoding the polypeptide; and 150 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT (b) a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that is at least 90% identical to any one of the sequences recited in TABLE 5. [573] Embodiment 2. The composition of embodiment 1, wherein the spacer sequence is at least 95% identical to any one of the sequences recited in TABLE 5. [574] Embodiment 3. The composition of embodiment 1, wherein the spacer sequence is at least 97% identical to any one of the sequences recited in TABLE 5. [575] Embodiment 4. The composition of embodiment 1, wherein the spacer sequence is at least 98% identical to any one of the sequences recited in TABLE 5. [576] Embodiment 5. The composition of embodiment 1, wherein the spacer sequence is at least 99% identical to any one of the sequences recited in TABLE 5. [577] Embodiment 6. The composition of embodiment 1, wherein the spacer sequence is identical to any one of the sequences recited in TABLE 5. [578] Embodiment 7. A composition that comprises: (a) a polypeptide or a nucleic acid encoding the polypeptide; and (b) a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a sgRNA that is at least 85% identical to any one of the sequences recited in TABLE 8. [579] Embodiment 8. The composition of embodiment 7, wherein the sgRNA is at least 90% identical to any one of the sequences recited in TABLE 8. [580] Embodiment 9. The composition of embodiment 7, wherein the sgRNA is at least 95% identical to any one of the sequences recited in TABLE 8. [581] Embodiment 10. The composition of embodiment 7, wherein the sgRNA is at least 99% identical to any one of the sequences recited in TABLE 8. [582] Embodiment 11. The composition of embodiment 7, wherein the sgRNA is identical to any one of the sequences recited in TABLE 8. [583] Embodiment 12. The composition of any one of embodiments 1-11, wherein the guide nucleic acid is a sgRNA in a single nucleic acid system and the sgRNA comprises a handle sequence and a spacer sequence. [584] Embodiment 13. The composition of embodiment 12, wherein the handle sequence is 5’ of the spacer sequence. 151 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [585] Embodiment 14. The composition of embodiment 12 or 13, wherein the guide nucleic acid comprises a handle sequence that is at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 100% identical to the sequence recited in TABLE 6. [586] Embodiment 15. A composition that comprises: (a) a polypeptide or a nucleic acid encoding the polypeptide; and (b) a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a crRNA that is at least 85% identical to any one of the sequences recited in TABLE 7. [587] Embodiment 16. The composition of embodiment 15, wherein the crRNA is at least 90% identical to any one of the sequences recited in TABLE 7. [588] Embodiment 17. The composition of embodiment 15, wherein the crRNA is at least 95% identical to any one of the sequences recited in TABLE 7. [589] Embodiment 18. The composition of embodiment 15, wherein the crRNA is at least 99% identical to any one of the sequences recited in TABLE 7. [590] Embodiment 19. The composition of embodiment 15, wherein the crRNA is identical to any one of the sequences recited in TABLE 7. [591] Embodiment 20. The composition of any one of embodiments 1-6, and 15-19, wherein the guide nucleic acid is a crRNA in a single nucleic acid system and the crRNA comprises a repeat sequence and a spacer sequence. [592] Embodiment 21. The composition of embodiment 20, wherein a repeat sequence is 5’ of the spacer sequence. [593] Embodiment 22. The composition of any one of embodiments 15-21, wherein the guide nucleic acid comprises a repeat sequence that is at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 100% identical to any one of the sequences recited in TABLE 4. [594] Embodiment 23. The composition of any one of embodiments 15-21, wherein the guide nucleic acid comprises a repeat sequence that is at least 90% identical to SEQ ID NO: 17. [595] Embodiment 24. The composition of any one of embodiments 15-21, wherein the guide nucleic acid comprises a repeat sequence that is at least 90% identical to SEQ ID NO: 18. [596] Embodiment 25. The composition of any one of embodiments 1-21 or 23, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 100% identical to SEQ ID NO: 1. 152 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [597] Embodiment 26. The composition of any one of embodiments 1-6, 15-21 or 24, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 100% identical to SEQ ID NO: 2. [598] Embodiment 27. The composition of any one of embodiments 1-21 or 23, wherein the polypeptide comprises a variant amino acid sequence of SEQ ID NO: 1, wherein the variant amino acid sequence comprises one or more amino acid alterations at one or more residues corresponding to one or more positions described in TABLE 1.1; and wherein the amino acid sequence, other than the one or more amino acid alterations has at least 90% sequence identity to the amino acid sequence referenced in SEQ ID NO: 1. [599] Embodiment 28. The composition of embodiment 27, wherein the one or more amino acid alteration is each independently a conservative or non-conservative substitution, or a combination thereof. [600] Embodiment 29. The composition of embodiment 28, wherein the one or more amino acid alterations are at one or more residues corresponding to one or more positions selected from: 58, 80, 84, 105, 193, 202, 209, 210, 218, 220, 225, 246, 286, 295, 298, 306, 315, and 360, or a combination thereof, relative to SEQ ID NO: 1. [601] Embodiment 30. The composition of embodiment 28, wherein the one or more amino acid alterations is each independently a substitution of an amino acid residue with a basic (positively charged) amino acid, and wherein the basic (positively charged) amino acid substitution is a substitution of an amino acid residue with a Lys (K), Arg (R), or His (H). [602] Embodiment 31. The composition of embodiment 30, wherein the one or more amino acid alterations are one or more amino acid substitutions selected from: D220R, E225R, A306K, N286K, E225K, I80K, S209F, Y315M, N193K, M298L, M295W, A306K, A218K, and K58W, or a combination thereof, relative to SEQ ID NO: 1. [603] Embodiment 32. The composition of embodiment 31, wherein the one or more amino acid alterations comprise a D220R substitution relative to SEQ ID NO: 1. [604] Embodiment 33. The composition of any one of embodiments 1-6, 15-21 or 24, wherein the polypeptide comprises a variant amino acid sequence of SEQ ID NO: 2, wherein the variant amino acid sequence comprises one or more amino acid alterations at one or more residues corresponding to one or more positions described in TABLE 1.1; and wherein the amino acid sequence, other than the one or more amino acid alterations has at least 90% sequence identity to the amino acid sequence referenced in SEQ ID NO: 2. [605] Embodiment 34. The composition of embodiment 33, wherein the one or more amino acid alteration is each independently a conservative or non-conservative substitution, or a combination thereof. [606] Embodiment 35. The composition of embodiment 33 or 34, wherein the one or more amino acid alterations are at one or more residues corresponding to one or more positions selected from: 2, 5, 11, 13, 153 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 51, 52, 53, 54, 55, 56, 57, 59, 68, 77, 79, 84, 87, 89, 90, 92, 94, 99, 100, 101, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 147, 149, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 220, 223, 231, 240, 258, 273, 276, 281, 285, 295, 301, 304, 312, 316, 329, 334, 340, 348, 355, 357, 363, 366, 370, 392, 399, 400, 405, 406, 407, 435, 445, 471, 480, 483, 497, 501, 503, 509, 511, 512, 513, 514, 515, 516, 517, 521, 523, 526, 529, 531, 536, 540, 541, 542, 543, 544, 545, 546, 549, 568, 577, 579, 590, 591, 592, 593, 594, 595, 596, 599, 602, 603, 604, 605, 606, 607, 608, 612, 617, 620, 624, 634, 638, 639, 653, 701, and 707, or a combination thereof, relative to SEQ ID NO: 2. [607] Embodiment 36. The composition of embodiment 35, wherein the one or more amino acid alterations is each independently each one or more amino acid residue alteration is independently a substitution with a basic (positively charged) amino acid, an acidic (negatively-charged) amino acid, a non- polar (hydrophobic) amino acid, or an uncharged polar amino acid, or a combination thereof. [608] Embodiment 37. The composition of embodiment 36, wherein the one or more amino acid alterations is each independently a substitution of an amino acid residue with an A, N, R, K, E, S, Q, P, T, G, F or D. [609] Embodiment 38. The composition of embodiment 37, wherein the one or more amino acid alterations are one or more amino acid substitutions selected from: A120R, A121Q, A130R, A24R, A35R, A366V, A602R, A606R, C193R, C285V, C357L, C363V, C36R, C405L, D113R, D501K, D512R, D523K, D549L, E100K, E101K, E109K, E109R, E119R, E258K, E31R, E33R, E34R, E42R, E44R, E529K, E536A, E595R, E68P, F14R, F202R, F312L, F39R, F445S, F509A, F53R, F701R, G111R, G122R, G136R, G13R, G179R, G25R, G276V, G32R, G497K, G55R, G56R, G577H, H110R, H208R, H20R, H604R, I2R, I126R, I127R, I17R, I191R, I203R, I240K, I471T, I59K, I603R, I653A, K118R, K128R, K135R, K15R, K184P, K184R, K189P, K200R, K206R, K29R, K348R, K37R, K38R, K392A, K99R, K281R, K407E, K435Q, K480L, K514R, K516R, K541R, K544R, K591R, K593R, K594R, K605R, K634G, K639E, K90E, K92E, K99S, L107F, L112R, L123R, L125R, L149R, L16R, L181R, L182R, L26R, L26K, L28R, L517R, L542R, L607F, L620E, M334E, M503K, M624A, N124R, N129R, N132R, N147K, N188R, N19R, N209R, N30R, N340S, N355R, N406K, N43R, N52R, N540R, N568D, N596R, P116G, P185R, P187I, P199R, P201R, P273A, P304E, P399F, P515R, P51R, P57R, P592R, P89T, P94E, P707R, Q138R, Q183R, Q195R, Q511R, Q54R, Q612R, Q79R, R18R, R22R, R329T, R41R, R531E, R546R, R617Y, S108R, S186G, S186R, S190R, S196R, S198R, S205R, S21R, S223P, S526N, S543R, S545R, S579R, S638K, S77V, T114R, T11R, T133R, T204R, T23R, T295N, T316R, T400L, T5R, T608R, T87G, V115R, V131R, V137R, V139R, V197R, V210R, V370L, V40R, V483G, V521T, V84Y, W599F, Y117R, Y134R, Y180R, Y192R, Y194R, Y207R, Y220S, Y231G, Y301L, Y513R, and Y590R, or a combination thereof, relative to SEQ ID NO: 2. 154 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [610] Embodiment 39. The composition of embodiment 38, wherein the one or more amino acid alteration is a L26R or L26K substitution relative to SEQ ID NO: 2. [611] Embodiment 40. The composition of any one of embodiments 27-39, wherein the variant polypeptide generates increased indels in a target nucleic acid relative to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2. [612] Embodiment 41. The composition of embodiment 40, wherein the variant polypeptide generates at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% more indels in a population of cells relative to the number of indels generated by a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2, as measured in a cleavage assay. [613] Embodiment 42. The composition of embodiment 27 or 33, wherein the polypeptide comprises at least one mutation that reduces its nuclease activity, relative to an otherwise comparable polypeptide without the mutation, as measured in a cleavage assay. [614] Embodiment 43. The composition of embodiment 42, wherein the variant polypeptide comprises one or more amino acid alterations, wherein the one or more amino acid alterations are one or more amino acid substitutions selected from: D237A, D418A, D418N, E335A, and E335Q, or a combination thereof, relative to SEQ ID NO: 1. [615] Embodiment 44. The composition of embodiment 42, wherein the variant polypeptide comprises one or more amino acid alterations, wherein the one or more amino acid alterations are one or more amino acid substitutions selected from: D369A, D369N, D658A, D658N, E567A, and E567Q, or a combination thereof, relative to SEQ ID NO: 2. [616] Embodiment 45. The composition of embodiment 43 or 44, wherein the variant polypeptide generates decreased indels in a target nucleic acid relative to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2. [617] Embodiment 46. The composition of embodiment 45, wherein the variant polypeptide generates about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, or about 1% less indels in a population of cells relative to the number of indels generated by a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, as measured in a cleavage assay. [618] Embodiment 47. The composition of any one of embodiments 42-46, wherein the polypeptide is catalytically inactive. [619] Embodiment 48. The composition of any one of embodiments 1-47, wherein the polypeptide is fused to one or more heterologous polypeptides. 155 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [620] Embodiment 49. The composition of embodiment 48, wherein the one or more heterologous polypeptides is fused to the N terminus, C terminus, or both of the polypeptide. [621] Embodiment 50. The composition of embodiment 48 or 49, wherein the one or more heterologous polypeptides is directly fused to the polypeptide by an amide bond or fused by at least one linker. [622] Embodiment 51. The composition of any one of embodiments 48-50, wherein the one or more heterologous polypeptides comprises a nuclear localization signal (NLS). [623] Embodiment 52. The composition of any one of embodiments 48-51, wherein the one or more heterologous polypeptides comprises any one of the sequences of TABLE 2. [624] Embodiment 53. The composition of any one of embodiments 48-50, wherein the one or more heterologous polypeptide is an effector partner. [625] Embodiment 54. The composition of embodiment 53, wherein the effector partner comprises a polypeptide selected from a deaminase, a transcriptional activator, a transcriptional repressor, or a functional domain thereof. [626] Embodiment 55. The composition of any one of embodiments 1-54, wherein the polypeptide recognizes a PAM sequence comprising any one of the sequences recited in TABLE 3, and wherein optionally the PAM sequence is adjacent to a target sequence of a target nucleic acid. [627] Embodiment 56. The composition of embodiment 55, wherein the target nucleic acid is a human DMPK gene. [628] Embodiment 57. The composition of embodiment 56, wherein the target nucleic acid comprises a mutation relative to the wild-type DMPK gene. [629] Embodiment 58. The composition of any one of embodiments 1-57, wherein the composition modifies one or more nucleotides of a target nucleic acid. [630] Embodiment 59. The composition of embodiment 58, wherein the modifying comprises cleaving at least one strand of a target nucleic acid, deleting or excising one or more nucleotides of the target nucleic acid, inserting one or more nucleotides into the target nucleic acid, substituting one or more nucleotides of the target nucleic acid with one or more alternative nucleotides, or a combination thereof. [631] Embodiment 60. The composition of embodiment 59, wherein the modifying comprises deleting or excising one or more nucleotides of a target nucleic acid. [632] Embodiment 61. The composition of any one of embodiments 1-60, wherein the composition comprises an additional engineered guide nucleic acid, or a nucleic acid encoding an additional engineered guide nucleic acid at least a portion of which hybridizes to a different target sequence of the target nucleic acid than the engineered guide nucleic acid. 156 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [633] Embodiment 62. The composition of embodiment 61, wherein the composition cleaves two loci of a target nucleic acid, and wherein the composition excises one or more nucleotides between the two loci of the target nucleic acid. [634] Embodiment 63. The composition of embodiment 59, wherein the modifying comprises deleting or excising one or more nucleotides of a target nucleic acid, wherein the one or more nucleotides are located in an untranslated region, protein coding region, an exon, an intron, a gene regulatory region, coding sequences thereof, or a combination thereof. [635] Embodiment 64. The composition of embodiment 63, wherein the one or more nucleotides are located in an untranslated region of a target nucleic acid. [636] Embodiment 65. The composition of embodiment 64, wherein the untranslated region is the 3’ untranslated region of a target nucleic acid. [637] Embodiment 66. The composition of embodiment 65, wherein the 3’ untranslated region of a target nucleic acid comprises a mutation comprising expansion of a (CTG)
n repeat. [638] Embodiment 67. The composition of embodiment 66, wherein the expansion of the (CTG)
n repeats are greater than about (CTG)
30, (CTG)
40, or about (CTG)
50. [639] Embodiment 68. The composition of embodiment 67, wherein the (CTG)
n repeats are about (CTG)
50 to about (CTG)
5,00. [640] Embodiment 69. The composition of any one of embodiments 1-68, wherein the mutation is associated with a disease. [641] Embodiment 70. The composition of embodiment 69, wherein the disease is any one of the diseases recited in TABLE 10. [642] Embodiment 71. The composition of embodiment 69, wherein the disease is myotonic dystrophy type 1. [643] Embodiment 72. A nucleic acid expression vector encodes a guide nucleic acid that comprises: (a) a spacer sequence that is at least 90% identical to any one of the sequences recited in TABLE 5, or a sgRNA that is at least 85% identical to any one of the sequences recited in TABLE 8; or (b) a spacer sequence that is at least 90% identical to any one of the sequences recited in TABLE 5, or a crRNA that is at least 85% identical to any one of the sequences recited in TABLE 7. [644] Embodiment 73. The nucleic acid expression vector of embodiment 72, wherein the nucleic acid expression vector is a viral vector. 157 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [645] Embodiment 74. The nucleic acid expression vector of embodiment 73, wherein the viral vector is an adeno associated viral (AAV) vector. [646] Embodiment 75. The nucleic acid expression vector of embodiment 73 or 74, wherein the viral vector comprises a nucleotide sequence of a first promoter, wherein the first promoter drives transcription of a nucleotide sequence encoding the guide nucleic acid, and wherein the first promoter is selected from a group consisting of CMV, EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GAL1-10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, CaMV35S, SV40, CMV, 7SK, and HSV TK. [647] Embodiment 76. The nucleic acid expression vector of any one of embodiments 72-75, wherein the nucleic acid expression vector comprises a nucleic acid sequence encoding a polypeptide that comprises an amino acid sequence that has at least 90% identity to any one of the sequences recited in TABLE 1, or a variant amino acid sequence thereof, wherein the variant amino acid sequence comprises one or more amino acid alterations, and wherein other than the one or more amino acid alterations the variant amino acid sequence has at least 80% sequence identity to any one of the sequences recited in TABLE 1. [648] Embodiment 77. The nucleic acid expression vector of embodiment 76, wherein the nucleic acid expression vector is a viral vector, wherein the viral vector comprises a nucleotide sequence of a second promoter, wherein the second promoter drives expression of the polypeptide, and wherein the second promoter is a ubiquitous promoter or a site-specific promoter. [649] Embodiment 78. The nucleic acid expression vector of embodiment 77, wherein the ubiquitous promoter is selected from a group consisting of MND and CAG. [650] Embodiment 79. The nucleic acid expression vector of embodiment 77, wherein the site-specific promoter is selected from a group consisting of Ck8e, Spc5-12, and Desmin. [651] Embodiment 80. The nucleic acid expression vector of any one of embodiments 72-79, wherein a viral vector comprises an enhancer, wherein the enhancer is a nucleotide sequence having the effect of enhancing promoter activity, wherein the enhancer is selected from a group consisting of WPRE enhancer, CMV enhancers, the R-U5′ segment in LTR of HTLV-I, SV40 enhancer, the intron sequence between exons 2 and 3 of rabbit β-globin, and the genome region of human growth hormone. [652] Embodiment 81. The nucleic acid expression vector of any one of embodiments 72-80, wherein the viral vector comprises a poly A signal sequence. [653] Embodiment 82. The nucleic acid expression vector of any one of embodiments 72-81, wherein the nucleic acid expression vector comprises a nucleotide sequence encoding a first guide nucleic acid and a nucleotide sequence encoding a second guide nucleic acid, and wherein the first guide nucleic acid is different from the second guide nucleic acid. [654] Embodiment 83. The nucleic acid expression vector of any one of embodiments 72-82 wherein the viral vector comprises a nucleotide sequence of a third promoter, wherein the third promoter drives 158 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT transcription of a nucleotide sequence encoding the second guide nucleic acid, wherein the third promoter is selected from a group consisting of CMV, EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GAL1-10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, CaMV35S, SV40, CMV, 7SK, and HSV TK, and wherein the first promoter and the third promoter are different. [655] Embodiment 84. The nucleic acid expression vector of embodiment 72 or 76, wherein at least one nucleic acid expression vector is a lipid or a lipid nanoparticle. [656] Embodiment 85. A pharmaceutical composition, comprising the composition of any one of embodiments 1-71 or the nucleic acid expression vector of any one of embodiments 72-84; and a pharmaceutically acceptable excipient, carrier or diluent. [657] Embodiment 86. A system comprising components for modification or detection of a target nucleic acid, wherein the components comprise: (a) a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that is at least 90% identical to any one of the sequences recited in TABLE 5; (b) any one of the compositions of embodiments 1-71, (c) the nucleic acid expression vector of any one of embodiments 72-84, or (d) the pharmaceutical composition of embodiment 85. [658] Embodiment 87. The system of embodiment 86, comprising at least one of: (a) a detection reagent; and (b) an amplification reagent. [659] Embodiment 88. The system of embodiment 87, wherein: (a) the detection reagent is selected from: a reporter nucleic acid, a detection moiety, and an additional polypeptide, or a combination thereof; and (b) the amplification reagent is selected from: a primer, a polymerase, a dNTP, and an rNTP, or a combination thereof. [660] Embodiment 89. The system of embodiment 87 or 88, wherein the detection reagent is operably linked to the polypeptide or the guide nucleic acid, such that a detection event occurs upon contacting the system with a target nucleic acid. [661] Embodiment 90. The system of any one of embodiments 87-89, wherein the amplification reagent amplifies a target nucleic acid. 159 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [662] Embodiment 91. A method of modifying a target nucleic acid in a cell, the method comprising contacting the cell with the composition of any one of embodiments 1-71, the nucleic acid expression vector of any one of embodiments 72-84, the pharmaceutical composition of embodiment 85, or the system of any one of embodiments 86-90, thereby modifying the target nucleic acid. [663] Embodiment 92. The method of embodiment 91, wherein the modifying of the target nucleic acid comprises cleaving at least one strand of a target nucleic acid, deleting or excising one or more nucleotides of the target nucleic acid, inserting one or more nucleotides into the target nucleic acid, substituting one or more nucleotides of the target nucleic acid with one or more alternative nucleotides, or a combination thereof. [664] Embodiment 93. The method of embodiment 91 or 92, wherein the modifying comprises deleting or excising one or more nucleotides of a target nucleic acid. [665] Embodiment 94. The method of any one of embodiments 91-93, wherein the method comprises the use of an additional engineered guide nucleic acid, or a nucleic acid encoding an additional engineered guide nucleic acid at least a portion of which hybridizes to a different target sequence of the target nucleic acid than the engineered guide nucleic acid. [666] Embodiment 95. The method of embodiment 94, wherein two loci of a target nucleic acid are cleaved and the one or more nucleotides between the two loci of the target nucleic acid are excised. [667] Embodiment 96. The method of embodiment 93 or 95, wherein the one or more nucleotides to be deleted or excised is an expansion of a (CTG)
n repeat in the 3’ UTR of a human DMPK gene. [668] Embodiment 97. The method of embodiment 96, wherein the expansion of the (CTG)
n is greater than about (CTG)
30. [669] Embodiment 98. The method of any one of embodiments 91-97, wherein the cell is ex vivo. [670] Embodiment 99. The method of any one of embodiments 91-97, wherein the cell is in vivo. [671] Embodiment 100. The method of any one of embodiments 91-99, wherein the cell is selected from an induced pluripotent stem cell (iPSC), a T cell, a hepatocyte, a cardiomyocyte, and a myoblast. [672] Embodiment 101. A cell contacted by the composition of any one of embodiments 1-71, the nucleic acid expression vector of any one of embodiments 72-84, the pharmaceutical composition of embodiment 85, the system of any one of embodiments 86-90, or the method of any one of embodiments 91-100. [673] Embodiment 102. A cell comprising the composition of any one of embodiments 1-71, the nucleic acid expression vector of any one of embodiments 72-84, the pharmaceutical composition of embodiment 85, or the system of any one of embodiments 86-90. [674] Embodiment 103. A cell that comprises a target nucleic acid modified by the composition of any one of embodiments 1-71, the nucleic acid expression vector of any one of embodiments 72-84, the 160 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT pharmaceutical composition of embodiment 85, the system of any one of embodiments 86-90, or the method of any one of embodiments 91-100. [675] Embodiment 104. The cell of any one of embodiments 102-103, wherein the cell is selected from an induced pluripotent stem cell (iPSC), a cardiomyocyte, and a myoblast. [676] Embodiment 105. A population of cells that comprises at least one cell of any one of embodiments 101-104. [677] Embodiment 106. A method of treating a disease associated with an expansion of a (CTG)
n repeat in the 3'-untranslated region (UTR) of a human DMPK gene in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of embodiment 85, or the system of embodiment 86. [678] Embodiment 107. The method of embodiment 106, wherein the disease is any one of the diseases recited in TABLE 9. [679] Embodiment 108. The method of embodiment 107, wherein the disease is myotonic dystrophy type 1. [680] Embodiment 109. The method of any one of embodiments 106-108, wherein the expansion of the (CTG)
n repeats are greater than about (CTG)
30, (CTG)
40, or about (CTG)
50. [681] Embodiment 110. The method of embodiment 109, wherein the (CTG)
n repeats are about (CTG)
50 to about (CTG)
5,000. 161 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT SEQUENCES AND TABLES [682] TABLE 1 provides illustrative amino acid sequences of effector proteins that are useful in the compositions, systems and methods described herein. TABLE 1. EXEMPLARY AMINO ACID SEQUENCE(S) OF EFFECTOR PROTEIN(S)
[683] TABLE 1.1 provides exemplary amino acid alterations relative to SEQ ID NO: 1 and SEQ ID NO: 2 useful in compositions, systems, and methods described herein. TABLE 1.1 EXEMPLARY AMINO ACID ALTERATIONS RELATIVE TO EFFECTOR PROTEIN(S)
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[684] TABLE 2 provides illustrative sequences of exemplary heterologous peptide modifications of effector protein(s) that are useful in the compositions, systems and methods described herein. TABLE 2. SEQUENCES OF EXEMPLARY HETEROLOGOUS PEPTIDE MODIFICATIONS
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[685] TABLE 3 provides illustrative PAM sequences that are useful in the compositions, systems and methods described herein. TABLE 3: PAM Sequences
*wherein each N is any nucleotide, each R is A or G, and each V is A, C or G. 169 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [686] TABLE 4 provides illustrative repeat sequences for use in guide nucleic acids that are useful in the compositions, systems and methods described herein. TABLE 4. EXEMPLARY REPEAT SEQUENCES FOR USE IN GUIDE NUCLEIC ACIDS
[687] TABLE 5 provides illustrative spacer sequences for use in guide nucleic acids that are useful in the compositions, systems and methods described herein. TABLE 5. EXEMPLARY SPACER SEQUENCES FOR USE IN GUIDE NUCLEIC ACIDS
[688] TABLE 6 provides illustrative handle sequences for use in guide nucleic acids that are useful in the compositions, systems and methods described herein, wherein all-caps no-formatting represents intermediary sequences, italic represents a linker, and underline represents repeat sequences. TABLE 6. EXEMPLARY HANDLE SEQUENCES FOR USE IN SINGLE GUIDE NUCLEIC ACID SYSTEMS 170 ACTIVE 700237712v1
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[689] TABLE 7 provides illustrative crRNA sequences that are useful in the compositions, systems and methods described herein, wherein italic represents repeat sequences and all-caps no-formatting represents the spacer sequence. TABLE 7. EXEMPLARY CRRNA SEQUENCES FOR USE IN SINGLE GUIDE NUCLEIC ACID SYSTEMS
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Attorney Docket No.203477-768601/PCT [690] TABLE 8 provides illustrative sgRNA sequences that are useful in the compositions, systems and methods described herein, wherein all-caps no-formatting represents intermediary sequences, italic represents a linker, underline represents repeat sequences, and bold represents the spacer sequence. TABLE 8. EXEMPLARY SGRNA SEQUENCES FOR USE IN SINGLE GUIDE NUCLEIC ACID SYSTEMS
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[691] TABLE 9 provides illustrative target nucleic acids that are useful in the compositions, systems and methods described herein. TABLE 9. EXEMPLARY TARGET NUCLEIC ACIDS
[692] TABLE 9.1 provides illustrative target nucleic acids that are useful in the compositions, systems and methods described herein. TABLE 9.1. EXEMPLARY TARGET NUCLEIC ACIDS
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[693] TABLE 9.2 provides genomic exonic and intronic locations of DMPK-201 as found at Ensembl No. ENST00000291270.9. TABLE 9.2. CERTAIN EXEMPLARY EXONIC AND INTRONIC LOCATIONS OF DMPK- 201
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[694] TABLE 10 provides illustrative diseases and syndromes for compositions, systems and methods described herein. TABLE 10. DISEASES AND SYNDROMES
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Attorney Docket No.203477-768601/PCT Parkinson Disease, Late-Onset; Myopathy, Centronuclear, 1; Hypothyroidism, Congenital, Nongoitrous, 2; Neuromyotonia and Axonal Neuropathy, Autosomal Recessive; Small Cell Cancer of the Lung; Ataxia with Vitamin E Deficiency; Thyrotropin-Releasing Hormone Deficiency; Ataxia-Telangiectasia; Hypothyroidism, Congenital, Nongoitrous, 4; Graves Disease 1; Mitochondrial Dna Depletion Syndrome 7; Hutterite Cerebroosteonephrodysplasia Syndrome; Frontotemporal Dementia; Hypertension, Essential; Attention Deficit-Hyperactivity Disorder; Neurodegeneration with Brain Iron Accumulation 2a; Papillon-Lefevre Syndrome; Batten-Turner Congenital Myopathy; Machado-Joseph Disease; Burkitt Lymphoma; Hypothyroidism, Congenital, Nongoitrous, 3; Aica-Ribosuria Due to Atic Deficiency; Hereditary Sensory Neuropathy; Cardiac Arrest; Basal Cell Carcinoma 1; Major Depressive Disorder; Ovarian Hyperstimulation Syndrome; Fragile X Syndrome; Fragile X Tremor/ataxia Syndrome; Spermatogenic Failure; Disease by Infectious Agent; Pulmonary Hypertension; Myofibrillar Myopathy; Hyperparathyroidism; Beriberi; Charcot-Marie-Tooth Disease, Dominant Intermediate B; Charcot-Marie-Tooth Disease, Axonal, Type 2e; Myotonia Congenita, Autosomal Recessive; Jawad Syndrome; Muscular Dystrophy-Dystroglycanopathy , Type a, 4; Premature Menopause; Hashimoto Thyroiditis; Tooth Disease; Charcot-Marie-Tooth Disease; Telangiectasis; Onchocerciasis; Graves' Disease; Leukemia; Endomyocardial Fibrosis; Wernicke Encephalopathy; Relapsing-Remitting Multiple Sclerosis; Colon Adenocarcinoma; Endometriosis; Hypothyroidism; Centronuclear Myopathy; Roussy-Levy Hereditary Areflexic Dystasia; Eating Disorder; Narcolepsy; Thymoma; Movement Disease; Hypokalemia; Nutritional Deficiency Disease; Neurofibromatosis; Osteoarthritis; Malignant Hyperthermia; Essential Tremor; Thyroiditis; Viral Infectious Disease; Episodic Ataxia; Encephalopathy; Periodic Paralysis; Syncope; Genetic Neuromuscular Disease; Twin-Reversed Arterial Perfusion Sequence; Metabolic Myopathy; Multiple Sclerosis; Neuropathy, Hereditary, with Liability to Pressure Palsies; Neurofibromatosis, Type I; Erythrocytosis, Familial, 1; Type 1 Diabetes Mellitus; Cystic Fibrosis; Camptodactyly-Arthropathy-Coxa Vara-Pericarditis Syndrome; Hypertriglyceridemia 1; Dentatorubral-Pallidoluysian Atrophy; Spinocerebellar Ataxia 1; Down Syndrome; Tuberous Sclerosis 1; Fetal Akinesia Deformation Sequence 1; Body Mass Index Quantitative Trait Locus 11; Hemophagocytic Lymphohistiocytosis, Familial, 1; Adrenoleukodystrophy; Myopathy, Centronuclear, X-Linked; Aging; Toe Syndactyly, Telecanthus, and Anogenital and Renal Malformations; Congenital Hemidysplasia with Ichthyosiform Erythroderma and Limb Defects; Charcot-Marie-Tooth Disease, X-Linked Dominant, 1; Agammaglobulinemia, X-Linked; Developmental and Epileptic Encephalopathy 1; Taqi Polymorphism; Hemophilia a; Rett Syndrome; Periodic Fever, Familial, Autosomal Dominant; Hypercholesterolemia, Familial, 1; Amyloidosis, Hereditary, Transthyretin-Related; Hypokalemic Periodic Paralysis, Type 1; Hyperkalemic Periodic Paralysis; Myelofibrosis; Nemaline Myopathy 2; Body Mass Index Quantitative Trait Locus 9; Body Mass Index Quantitative Trait Locus 8; Dermatitis, Atopic; Fanconi Anemia, Complementation Group E; Gallbladder Disease 1; Pulmonary Alveolar Proteinosis, Acquired; Muscular Dystrophy, Limb-Girdle, Autosomal Recessive 4; Distal Arthrogryposis; Diffuse Large B-Cell Lymphoma; Atrioventricular Block; Body Mass Index Quantitative Trait Locus 18; Tatton-Brown-Rahman Syndrome; Scoliosis; Non- Alcoholic Fatty Liver Disease; Miyoshi Muscular Dystrophy; Chromosomal Disease; Myopathy, Myofibrillar, 4; Body Mass Index Quantitative Trait Locus 12; Body Mass Index Quantitative Trait Locus 4; Pulmonary Disease, Chronic Obstructive; Leukemia, Chronic Myeloid; Body Mass Index Quantitative Trait Locus 14; Body Mass Index Quantitative Trait Locus 7; Body Mass Index Quantitative Trait Locus 10; Meningioma, Familial; Prostate Cancer, Hereditary, 8; Leukemia, Acute Myeloid; Neurofibromatosis-Noonan Syndrome; Cardiomyopathy, Dilated, 1b; Myopathy, Myofibrillar, 1; Carnitine Palmitoyltransferase Ii Deficiency, Infantile; Brody Disease; Suppression of Tumorigenicity 12; Chylothorax, Congenital; Cdags Syndrome; Cholelithiasis; Foodborne Botulism; Parkinsonism; Sleeping Sickness; Trypanosomiasis; Cerebellar Atrophy, Developmental Delay, and Seizures; Body Mass Index Quantitative Trait Locus 19; Helix Syndrome; Body Mass Index Quantitative Trait Locus 20; Prostate Cancer, Hereditary, 6; Leukemia, Acute Lymphoblastic; Ewing Sarcoma; Fatty Liver Disease, Nonalcoholic 1; Renal Cell Carcinoma, Nonpapillary; Neurofibromatosis, Type Ii; Ankyloglossia with or Without Tooth Anomalies; Hyperparathyroidism 1; Central Core Disease of Muscle; Charcot-Marie-Tooth Disease, Demyelinating, Type 1a; Chopra-Amiel-Gordon Syndrome; Sick Sinus Syndrome; Neonatal 176 ACTIVE 700237712v1
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Attorney Docket No.203477-768601/PCT EXAMPLES [695] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Example 1. Indel activity of Effector Proteins (paired with Single Guide Nucleic Acid) in Eukaryotic Cells [696] Effector proteins (e.g., any one of the effector proteins recited in TABLE 1, or a variant thereof as recited in TABLE 1.1) are tested for their ability to produce indels at the targeted loci (e.g., DMPK gene) in eukaryotic cells (e.g., induced pluripotent stem cell (iPSC), a T cell, a hepatocyte, a cardiomyocyte, a myoblast, a HEK293T cell, a muscle cell, or any other eukaryotic cell) in vitro. Plasmid pairs co-expressing the effector protein and a single guide nucleic acid (e.g., guide RNA, sgRNA or crRNA) are delivered to eukaryotic cells via transfection, electroporation, or lipofection using a lipofection reagent. Transfected cells are first incubated with the effector protein and guide nucleic acid and then lysed to obtain the genomic DNA. The genomic DNA is subjected to PCR amplification to amplify the targeted loci. Indels are detected by next generation sequencing (NGS) of PCR amplicons at the targeted loci, and indel percentage is calculated as the fraction of sequencing reads containing insertions or deletions relative to an unedited reference sequence. Example 2. Indel activity of Effector Proteins (paired with Dual Nucleic Acid) in Eukaryotic Cells [697] Effector proteins (e.g., any one of the effector proteins recited in TABLE 1, or a variant thereof as recited in TABLE 1.1) are tested for their ability to produce indels at the targeted loci (e.g., DMPK gene) in eukaryotic cells (e.g., induced pluripotent stem cell (iPSC), a T cell, a hepatocyte, a cardiomyocyte, a myoblast, a HEK293T cell, a muscle cell, or any other eukaryotic cell) in vitro. Plasmid pairs co-expressing the effector protein and a dual nucleic acid (e.g., guide RNA, sgRNA or crRNA) are delivered to eukaryotic cells via transfection, electroporation, or lipofection using a lipofection reagent. Transfected cells are first incubated with the effector protein and dual nucleic acid and then lysed to obtain the genomic DNA. The genomic DNA is subjected to PCR amplification to amplify the targeted loci. Indels are detected by next generation sequencing (NGS) of PCR amplicons at the targeted loci, and indel percentage is calculated as the fraction of sequencing reads containing insertions or deletions relative to an unedited reference sequence. Example 3. AAV vectors comprising Effector Protein [698] An AAV vector is constructed to contain a transgene between its ITRs. The transgene provides or encodes, in a 5’ to 3’ direction, a first promoter, a guide nucleic acid (e.g., sgRNA and/or crRNA, a first guide nucleic acid), a second promoter, an effector protein (e.g., a variant of any one of the sequence recited in TABLE 1) as described herein, and a poly A signal. Optionally, the AAV vector 178 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT comprises additional promoters (e.g., a third promoter driving the transcription of a second guide nucleic acid), guide nucleic acids (e.g., a second guide nucleic acid of a dual nucleic acid system), transcriptional enhancer(s) (e.g., WPRE enhancer, CMV enhancer(s), R-U5′ segment in LTR of HTLV- I, SV40 enhancer, intron sequence between exons 2 and 3 of rabbit β-globin, or genome region of human growth hormone), or combinations thereof. Additional guide nucleic acids comprise different spacer sequences targeting different sequences in a target nucleic acid (e.g., DMPK gene). Additionally, the effector protein can be capable of editing the target sequence such that contact of the effector protein- guide nucleic acid complex to the target nucleic acid results in editing of a target sequence (e.g., deletion of the (CTG)
n repeat region of the DMPK gene). The effector protein can be expressed either ubiquitously or site-specifically (e.g., tissue-specifically) based on the second promoter the AAV vector is engineered to have. The first AAV vector promoter can be independently selected from a group consisting of CMV, EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GAL1-10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, CaMV35S, SV40, CMV, 7SK, and HSV TK. The second AAV vector promoter can be a ubiquitous promoter independently selected from a group consisting of MND and CAG. Alternatively, the second AAV vector promoter can be a site- specific promoter independently selected from a group consisting of Ck8e, Spc5-12, and Desmin. Optionally, the AAV vector comprises an additional promoter and an additional guide nucleic acid, such that the additional promoter is a third promoter independently selected from a group consisting of CMV, EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GAL1-10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, CaMV35S, SV40, CMV, 7SK, and HSV TK. However, the first promoter and the third promoter are different. The Poly A signal sequence is either hGH Poly A signal sequence or sv40 Poly A signal sequence. The AAV vector is expressed with supporting plasmids to produce an adeno-associated virus (AAV). Example 4. Gene editing of Eukaryotic Cells with AAV vector comprising Effector Protein [699] An AAV vector is constructed to contain a transgene between its ITRs. Such a transgene, for example, can provide or encode, in a 5’ to 3’ direction, a first promoter (e.g., U6), a guide nucleic acid (e.g., guide RNA, sgRNA, crRNA), a second promoter (e.g., MND, CAG, Ck8e, Spc5-12, Desmin), an effector protein (e.g., any one of the effector proteins recited in TABLE 1, or a variant thereof as recited in TABLE 1.1), and a poly A signal (e.g., SV40 poly A tail). Optionally, the AAV vector comprises additional promoters, guide nucleic acids, transcriptional enhancers, or combinations thereof. The AAV vector is expressed with supporting plasmids to produce an adeno-associated virus (AAV). Eukaryotic cells (e.g., induced pluripotent stem cell (iPSC), a T cell, a hepatocyte, a cardiomyocyte, a myoblast, a HEK293T cell, a muscle cell, or any other eukaryotic cell) are contacted with the AAV for 24 hours. After about 96 hours, post AAV contact, DNA or RNA is isolated from the infected eukaryotic cells. Edits are detected at the targeted loci (e.g., DMPK gene) by next generation sequencing (NGS) and/or Q-PCR. 179 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT Example 5. Gene editing of Eukaryotic Cells with AAV vector comprising Fusion Proteins [700] An AAV vector is constructed to contain a transgene between its ITRs. In some embodiments, the transgene providing or encoding, in a 5’ to 3’ direction, a first promoter (e.g., U6), a guide nucleic acid (e.g., guide RNA, sgRNA, crRNA), a second promoter (e.g., MND, CAG, Ck8e, Spc5-12, Desmin), an fusion protein (e.g., effector protein - fusion protein comprises an effector protein or a variant thereof (e.g., TABLE 1 and TABLE 1.1) fused to an effector partner), and a poly A signal (e.g., SV40 poly A tail). Optionally, the AAV vector comprises additional promoters, guide nucleic acids, transcriptional enhancers, or combinations thereof. The AAV vector is expressed with supporting plasmids to produce an adeno-associated virus (AAV). Eukaryotic cells (e.g., induced pluripotent stem cell (iPSC), a T cell, a hepatocyte, a cardiomyocyte, a myoblast, a HEK293T cell, a muscle cell, or any other eukaryotic cell) are contacted with the AAV for 24 hours. After about 96 hours, post AAV contact, DNA or RNA is isolated from the infected eukaryotic cells. Edits are detected at the targeted loci (e.g., DMPK gene) by next generation sequencing (NGS) and/or Q-PCR. Example 6. CasM.265466 mediated DMPK gene modification in HEK293T cells [701] Exemplary guide nucleic acids (e.g., sgRNA) targeting the gene encoding myotonic dystrophy protein kinase (i.e, DMPK) were screened in eukaryotic cells (e.g., HEK293T cells) for the identification and selection of guide nucleic acids for gene modification therapeutic strategies. [702] The DMPK gene has a UTR sequence containing repeats of (CTG)
n downstream of the coding region of Exon 14, which can vary in the number of repeats, often expanding from one generation to the next and varying among cell types (e.g., muscle cells can have even higher repeats). When the repeat region exceeds a certain number (typically about 50 repeats), transcription of the DMPK gene into RNA can result in the RNA transcript becoming toxic, causing the disease myotonic dystrophy type 1 (DM1). [703] Using the CasM.265466 as described in TABLE 1 as the effector protein, the present assay targets the (CTG)n repeat region of the DMPK gene. [704] Briefly, guide RNAs were designed to have a handle sequence as described in TABLE 6 followed by a 20nt spacer sequence as described in TABLE 5. The handle sequence containing an intermediary RNA sequence, a GAAA linker sequence, and a 13nt repeat sequence as described in TABLE 4. The resulting guide RNA with a 5’ to 3’ structure as described in TABLE 8 (e.g., 5’- intermediary RNA – GAAA – AAGGAUGCCAAAC – SPACER – 3’ (SEQ ID NO: 173)). Replicates (Rep 1 or Rep 2) of plasmid pairs co-expressing the effector protein and a single guide RNA system or dual nucleic acid system, as described in TABLE 11, were delivered to HEK293T cells via lipofection using a lipofection reagent. 180 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT TABLE 11. EXEMPLARY CASM.265466 AND GUIDE RNA COMPOSITIONS
[705] Plasmids encoding the single guide RNA system were screened to establish general modification activity, while plasmids encoding the dual nucleic acid system were screened to establish modification/region deletion activity. Both single and dual nucleic acid systems were assessed in batches of two replicates (Rep 1 and Rep 2), as shown in TABLE 12. [706] Lipofected cells were first incubated with the effector protein and guide RNA for 72 hours before being lysed and subjected to PCR amplification. Indels and region deletions were detected by next generation sequencing (NGS) of PCR amplicons at the targeted loci. Briefly, percentage modified was calculated as the fraction of sequencing reads containing insertions or deletions relative to an unmodified reference sequence, and percentage full region deletion was calculated as the fraction of sequencing reads containing full region deletions relative to an unmodified reference sequence. [707] Results demonstrating activity of CasM.265466 effector protein and the single guide RNA system are shown in TABLE 12. Results demonstrating activity of CasM.265466 effector protein and the dual nucleic acid system are shown in TABLE 13. The results demonstrate that the CasM.265466 effector protein is capable of deleting the (CTG)
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Attorney Docket No.203477-768601/PCT TABLE 12. CASM.265466 AND SINGLE GUIDE RNA SYSTEM (COMPOSITIONS 1-7 FROM TABLE 11)
TABLE 13. CASM.265466 AND DUAL NUCLEIC ACID SYSTEM (COMPOSITIONS 8-17 FROM TABLE 11)
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Attorney Docket No.203477-768601/PCT Example 7. CasPhi.12 L26R Variant mediated DMPK gene modification in HEK293T cells [708] Exemplary guide RNAs (e.g., crRNA) targeting the gene encoding myotonic dystrophy protein kinase (i.e, DMPK) were screened in eukaryotic cells (e.g., HEK293T cells) for the identification and selection of guide nucleic acids for gene modification therapeutic strategies. [709] The DMPK gene has a UTR sequence containing repeats of (CTG)
n downstream of the coding region of Exon 14, which can vary in the number of repeats, often expanding from one generation to the next and varying among cell types (e.g., muscle cells can have even higher repeats). When the repeat region exceeds a certain number (typically about 50 repeats), transcription of the DMPK gene into RNA can result in the RNA transcript becoming toxic, causing the disease myotonic dystrophy type 1 (DM1). [710] Using the CasPhi.12 L26R Variant (the L26R Variant with respect to SEQ ID NO: 2) as the effector protein, the present assay targets the (CTG)
n repeat region of the DMPK gene. [711] Briefly, guide RNAs were designed to have a 24nt repeat sequence (AUAGAUUGCUCCUUACGAGGAGAC (SEQ ID NO: 18)) for binding by the CasPhi.12 L26R Variant followed by a 20nt spacer sequence as described in TABLE 14. [712] In batches of two replicates (Rep 1 and Rep 2), plasmids encoding a single guide RNA system or a dual nucleic acid system were generated, normalized to 100ng/uL, and mixed to a ratio of 1:1 with 100ng/uL of plasmids encoding the L26R Variant. Plasmids were delivered to HEK293T cells via lipofection using a lipofection reagent. TABLE 14. L26R Variant (relative to SEQ ID NO: 2)- GUIDE RNA COMPOSITIONS
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[713] Plasmids encoding the single guide RNA system were screened to establish general modification activity, while plasmids encoding the dual nucleic acid system were screened to establish modification/region deletion activity. Both single and dual nucleic acid systems were assessed in batches of two replicates (Rep 1 and Rep 2), as shown in TABLE 14. [714] Lipofected cells were incubated for 72 hours before being harvested for DNA, PCR amplified and sequenced via next generation sequencing (NGS). The sequencing data was analyzed to detect/quantify % indel and region deletions. [715] Results demonstrating activity of L26R variant effector protein and the single guide RNA system activity are indicated in TABLE 15 and FIG.1. Results demonstrating activity of L26R variant effector protein and the dual nucleic acid system are shown in TABLE 16 and FIG. 2. The results demonstrate that the L26R variant is capable of deleting the repeat region of DMPK gene (CTG)
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Attorney Docket No.203477-768601/PCT TABLE 15. L26R Variant (relative to SEQ ID NO: 2) AND SINGLE GUIDE RNA SYSTEM (COMPOSITIONS 18-33 FROM TABLE 14)
TABLE 16. L26R Variant (relative to SEQ ID NO: 2) AND DUAL NUCLEIC ACID SYSTEM (COMPOSITIONS 34-59 FROM TABLE 14)
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[683] TABLE 1.1 provides exemplary amino acid alterations relative to SEQ ID NO: 1 and SEQ ID NO: 2 useful in compositions, systems, and methods described herein. TABLE 1.1 EXEMPLARY AMINO ACID ALTERATIONS RELATIVE TO EFFECTOR PROTEIN(S)
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[684] TABLE 2 provides illustrative sequences of exemplary heterologous peptide modifications of effector protein(s) that are useful in the compositions, systems and methods described herein. TABLE 2. SEQUENCES OF EXEMPLARY HETEROLOGOUS PEPTIDE MODIFICATIONS
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[685] TABLE 3 provides illustrative PAM sequences that are useful in the compositions, systems and methods described herein. TABLE 3: PAM Sequences
*wherein each N is any nucleotide, each R is A or G, and each V is A, C or G. 169 ACTIVE 700237712v1
Attorney Docket No.203477-768601/PCT [686] TABLE 4 provides illustrative repeat sequences for use in guide nucleic acids that are useful in the compositions, systems and methods described herein. TABLE 4. EXEMPLARY REPEAT SEQUENCES FOR USE IN GUIDE NUCLEIC ACIDS
[687] TABLE 5 provides illustrative spacer sequences for use in guide nucleic acids that are useful in the compositions, systems and methods described herein. TABLE 5. EXEMPLARY SPACER SEQUENCES FOR USE IN GUIDE NUCLEIC ACIDS
[688] TABLE 6 provides illustrative handle sequences for use in guide nucleic acids that are useful in the compositions, systems and methods described herein, wherein all-caps no-formatting represents intermediary sequences, italic represents a linker, and underline represents repeat sequences. TABLE 6. EXEMPLARY HANDLE SEQUENCES FOR USE IN SINGLE GUIDE NUCLEIC ACID SYSTEMS 170 ACTIVE 700237712v1
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[689] TABLE 7 provides illustrative crRNA sequences that are useful in the compositions, systems and methods described herein, wherein italic represents repeat sequences and all-caps no-formatting represents the spacer sequence. TABLE 7. EXEMPLARY CRRNA SEQUENCES FOR USE IN SINGLE GUIDE NUCLEIC ACID SYSTEMS
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Attorney Docket No.203477-768601/PCT [690] TABLE 8 provides illustrative sgRNA sequences that are useful in the compositions, systems and methods described herein, wherein all-caps no-formatting represents intermediary sequences, italic represents a linker, underline represents repeat sequences, and bold represents the spacer sequence. TABLE 8. EXEMPLARY SGRNA SEQUENCES FOR USE IN SINGLE GUIDE NUCLEIC ACID SYSTEMS
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[691] TABLE 9 provides illustrative target nucleic acids that are useful in the compositions, systems and methods described herein. TABLE 9. EXEMPLARY TARGET NUCLEIC ACIDS
[692] TABLE 9.1 provides illustrative target nucleic acids that are useful in the compositions, systems and methods described herein. TABLE 9.1. EXEMPLARY TARGET NUCLEIC ACIDS
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[693] TABLE 9.2 provides genomic exonic and intronic locations of DMPK-201 as found at Ensembl No. ENST00000291270.9. TABLE 9.2. CERTAIN EXEMPLARY EXONIC AND INTRONIC LOCATIONS OF DMPK- 201
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[694] TABLE 10 provides illustrative diseases and syndromes for compositions, systems and methods described herein. TABLE 10. DISEASES AND SYNDROMES
TABLE 13. CASM.265466 AND DUAL NUCLEIC ACID SYSTEM (COMPOSITIONS 8-17 FROM TABLE 11)
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Attorney Docket No.203477-768601/PCT Example 7. CasPhi.12 L26R Variant mediated DMPK gene modification in HEK293T cells [708] Exemplary guide RNAs (e.g., crRNA) targeting the gene encoding myotonic dystrophy protein kinase (i.e, DMPK) were screened in eukaryotic cells (e.g., HEK293T cells) for the identification and selection of guide nucleic acids for gene modification therapeutic strategies. [709] The DMPK gene has a UTR sequence containing repeats of (CTG)n downstream of the coding region of Exon 14, which can vary in the number of repeats, often expanding from one generation to the next and varying among cell types (e.g., muscle cells can have even higher repeats). When the repeat region exceeds a certain number (typically about 50 repeats), transcription of the DMPK gene into RNA can result in the RNA transcript becoming toxic, causing the disease myotonic dystrophy type 1 (DM1). [710] Using the CasPhi.12 L26R Variant (the L26R Variant with respect to SEQ ID NO: 2) as the effector protein, the present assay targets the (CTG)n repeat region of the DMPK gene. [711] Briefly, guide RNAs were designed to have a 24nt repeat sequence (AUAGAUUGCUCCUUACGAGGAGAC (SEQ ID NO: 18)) for binding by the CasPhi.12 L26R Variant followed by a 20nt spacer sequence as described in TABLE 14. [712] In batches of two replicates (Rep 1 and Rep 2), plasmids encoding a single guide RNA system or a dual nucleic acid system were generated, normalized to 100ng/uL, and mixed to a ratio of 1:1 with 100ng/uL of plasmids encoding the L26R Variant. Plasmids were delivered to HEK293T cells via lipofection using a lipofection reagent. TABLE 14. L26R Variant (relative to SEQ ID NO: 2)- GUIDE RNA COMPOSITIONS
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TABLE 16. L26R Variant (relative to SEQ ID NO: 2) AND DUAL NUCLEIC ACID SYSTEM (COMPOSITIONS 34-59 FROM TABLE 14)
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