Attorney Docket No.: 063586-537001WO CHEMICAL MODIFICATIONS OF GUIDE RNAS FOR CRISPR NUCLEASES CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Application No.63/575,058, filed April 5, 2024, the entire contents of which is incorporated by reference herein. SEQUENCE LISTING The instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 26, 2024, is named “063586-537001WO_Sequence_Listing- v3.xml” and is 907,386 bytes in size. BACKGROUND OF THE INVENTION Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR- associated (Cas) genes, collectively known as CRISPR-Cas or CRISPR/Cas systems, are adaptive immune systems in archaea and bacteria that defend particular species against foreign genetic elements. A CRISPR-Cas system typically comprises a CRISPR nuclease and one or more RNA components that direct the CRISPR nuclease toa target genomic site for gene editing. It is of interest to develop efficient CRISPR gene editing systems to improve gene editing efficiency and accuracy. SUMMARY OF THE INVENTION The present disclosure is based, at least in part, on the development of specific modification patterns of guide RNAs for a Type V CRISPR nuclease designed based on structural analysis of the Type V CRISPR nuclease complexed with its cognate guide RNA. Guide RNAs with such designed modification patterns, in association with the Type V CRISPR nuclease, resulted in enhanced on-target editing efficiencies and reduced off-target editing efficiencies. Gene editing systems comprising the Type V CRISPR nuclease and the modified guide RNAs as provided herein also showed desirable in vivo editing efficiency at target gene sites. Accordingly, provided herein are guide RNAs having a specific modification pattern, gene editing systems comprising such a modified guide RNA and a Type V CRISPR nuclease
Attorney Docket No.: 063586-537001WO (e.g., those provided herein), and gene editing methods involving the use of such gene editing systems. In some aspects, the present disclosure features a modified guide RNA comprising a structure of 5’-N1-N2-DR-SP-3’ (Formula I), in which each of N1 and N2 independently is absent or a nucleotide; DR is a direct repeat sequence recognizable by a Type V CRISPR nuclease; and SP is a spacer sequence specific to a target sequence in a genomic site. The target sequence is 3’ to a protospacer-adjacent motif (PAM) of 5’-NTTR-3’, in which N is A, T, G, or C, and R is A or G. Up to three nucleotides at the 5’ end of the guide RNA have 2’- O-methylation, phosphorothioate bond modification, or a combination thereof. Alternatively or in addition, up to three nucleotides at the 3’ end of the guide RNA have 2’-O-methylation, phosphorothioate bond modification, or a combination thereof. Further, the 2’ position of the second nucleotide, the third nucleotide, or both at the 5’ end of the DR are unmodified (i.e., having an -OH group at the 2’ position). In some embodiments, both N1 and N2 are present in Formula I. In some examples, N1 can be G. Alternatively or in addition, N2 can be A. In other embodiments, both N1 and N2 are absent. In some embodiments, the three nucleotides at the 3’ end of the guide RNA have both 2’-O-methylation and phosphorothioate bond modifications. Alternatively or in addition, one, two, or three nucleotides at the 5’ end of the guide RNA have a phosphorothioate bond modification(s). In some examples, the 5’ end three nucleotides have phosphorothioate bond modifications. In other examples, the 5’ end nucleotide has a phosphorothioate bond modification. In yet other examples, the 5’ end two nucleotides have 2’-O-methylation and phosphorothioate bond modifications. In specific examples, the modified guide RNA has the modification pattern of mod2, mod4, mod6, or mod7 provided herein. In one specific example, the modified guide RNA has the mod4 modification pattern. In any of the modified guide RNAs disclosed herein, the DR comprises a nucleotide sequence at least 90% identical to 5'-CUUGUUGUAUAUGUCCUUUUAUAGGUAUUAA ACAAC-3' (SEQ ID NO: 7). In one example, the DR is set forth as SEQ ID NO: 7. In some embodiments, the spacer sequence (SP) is 15-25-nt in length, for example, 16-20-nt in length, or 16-18-nt in length. In one example, the spacer sequence is 16-nt in length. In another example, the spacer sequence is 18-nt in length. In yet another example, the spacer sequence is 20-nt in length. As reported herein, shorter spacers exhibited better gene editing efficacy and decreased off-target editing. In some instances, the shorter spacers have a 3’ end truncation
Attorney Docket No.: 063586-537001WO (e.g., up to 5-nucleotides, such as 4-nt, 3-nt, 2-nt or 1nt) relative to the counterpart spacers that have the same length as the target sequence (e.g., 20-nt in length). Such truncated versions of any of the specific spacer sequences disclosed herein are within the scope of the present disclosure. In other aspects, the present disclosure features a gene editing system, comprising: (a) a Type V CRISPR nuclease or a nucleic acid encoding the Type V CRISPR nuclease polypeptide; and (b) a modified guide RNA as those disclosed herein. In some embodiments, the Type V CRISPR nuclease polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1. In some examples, the Type V CRISPR nuclease polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, the Type V CRISPR nuclease polypeptide comprises one or more mutations relative to SEQ ID NO: 1. In some instances, the one or more mutations are amino acid substitutions, which optionally are at positions P14, D32, I61, E311, T338, and/or E736 in SEQ ID NO: 1, for example, P14R, D32R, I61R, E311R, T338G, and/or E736G relative to SEQ ID NO: 1. In one example, the Type V CRISPR nuclease polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In another example, the Type V CRISPR nuclease polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In some instances, the Type V CRISPR nuclease polypeptide is a fusion polypeptide further comprising one or more functional elements, which are heterologous to the CRISPR nuclease moiety therein. In some examples, the one or more functional elements comprises one or more nuclear localization signals (NLSs), one or more peptide linkers, or a combination thereof. In some embodiments, the gene editing system provided herein may comprise the Type V CRISPR nuclease polypeptide. In some instances, the Type V CRISPR nuclease polypeptide and the modified guide RNA form a ribonucleoprotein (RNP) complex. Alternatively, the gene editing system may comprise a nucleic acid encoding the Type V CRISPR nuclease polypeptide. In some examples, the nucleic acid may be a messenger RNA (mRNA). Any of the gene editing systems provided herein may further comprise one or more lipid excipients associated with element (a) (the Type V CRISPR nuclease polypeptide or its encoding nucleic acid) and/or element (b) (the modified guide RNA). In some examples, the one or more lipid excipients form lipid nanoparticles. Also provided herein is a pharmaceutical composition comprising any of the gene editing systems disclosed herein. Further, the instant disclosure provides a kit comprising (a)
Attorney Docket No.: 063586-537001WO the Type V CRISPR nuclease or the nucleic acid encoding the Type V CRISPR nuclease set forth herein; and (b) the modified guide RNA also set forth herein. In yet other aspects, the present disclosure features a method for editing a gene in a cell, the method comprising contacting a host cell with the gene editing system disclosed herein to allow for genetic editing of the gene by the gene editing system. The spacer sequence in the modified guide RNA of the gene editing system is specific to a target sequence within the gene. The target sequence is 3’ to a protospacer-adjacent motif (PAM) of 5’-NTTR-3’, in which N is A, T, G, or C, and R is A or G. In some instances, the host cell is cultured in vitro. In other instances, the contact step is performed by delivering the gene editing system to a subject comprising the host cell. Also within the scope of the present disclosure is a gene editing system as disclosed herein for use in editing a target gene, as well as use of such a gene editing system for manufacturing a medicament for use in editing a target gene in a subject to achieve therapeutic purposes. The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein. FIG.1 is a diagram showing PCSK9 indels in a PHH donor following electroporation of the CRISPR nuclease (mRNA or polypeptide) and the RNA guide (unmodified or both ends modified). For each sample, from left to right, unmodified gRNA/nuclease protein, both ends modified gRNA/nuclease, unmodified gRNA/nuclease mRNA; and both ends modified gRNA/nuclease mRNA. FIGs.2A-2E include diagrams showing predicted nuclease structure, modification patterns of guide RNAs designed thereby, and gene editing efficiencies using the modified guide RNAs. FIG.2A: a diagram illustrating the predicted structure of the nuclease/gRNA complex. FIG.2B: PCSK9 or HAO1 gene editing efficiency using a modified gRNA as indicated and an mRNA molecule encoding the CRISPR nuclease polypeptide of SEQ ID
Attorney Docket No.: 063586-537001WO NO: 6. FIG.2C: PCSK9 or HAO1 gene editing efficiency using an RNP complex comprising a modified gRNA as indicated and the CRISPR nuclease polypeptide of SEQ ID NO: 6. FIG.2D: PCSK9 or HAO1 gene editing efficiency using mod2, mod4, or mod6 gRNA as indicated and an mRNA molecule encoding the CRISPR nuclease polypeptide of SEQ ID NO: 6. FIG.2E: PCSK9 or HAO1 gene editing efficiency using an RNP complex comprising mod2, mod4, or mod6 gRNA as indicated and the CRISPR nuclease polypeptide of SEQ ID NO: 6. FIGS.3A-3B are diagrams showing indels at PCSK9 target site in a PHH donor (HU2186, Thermofisher) following electroporation of mRNA effector with RNA guides containing either 16 nt, 18 nt, or 20 nt spacers for PCSK9-E1T3 (FIG.3A) or PCSK9-E10T8 (FIG.3B). Each dot represents the average of three technical replicates across one biological replicate. Error bars represent standard deviation. The data shows higher on-target activity for 18 nt compared to 16 nt and 20 nt spacers. FIGS.4A-4E are diagrams showing indels at in silico predicted off-target sites in a PHH donor (HU2186, Thermofisher) following electroporation of mRNA effector with RNA guides containing either 16 nt, 18 nt, or 20 nt spacers for PCSK9-E1T3 (FIGS.4A-4B) or PCSK9-E10T8 (FIGS.4C-4E). Each dot represents the average of three technical replicates across one biological replicate. Error bars represent standard deviation. The data shows higher off-target activity for 18 nt compared to 16 nt and 20 nt spacers, and elimination of off-target activity at sites tested for the 16 nt spacer length guides. FIG.5 is a diagram showing interim PCSK9 levels in genetically edited mice. FIGs.6A-6C include diagrams showing in vivo genetic editing of HAO1 in mouse liver cells using the CRISPR nuclease polypeptide of SEQ ID NO: 6 and guide RNAs with mod2, mod4, or mod7 modifications. DETAILED DESCRIPTION OF THE INVENTION Guide RNAs used in CRISPR nuclease-mediated gene editing methods often contain modifications to enhance stability of the RNA when delivered to host cells. Surprisingly, it is reported herein that 2’-O-methylation and phosphorothioate bond modifications at both 5’ and 3’ ends of a guide RNA reduced gene editing activities by a cognate Type V CRISPR nuclease, as relative to the unmodified counterpart. Structural analysis of the Type V CRISPR nuclease complexed with a cognate guide RNA shows that the 2’ hydroxyl group of the 2nd and 3rd nucleotides at the 5’ end of the direct repeat (DR) sequence in the guide RNA are involved in the interaction with the CRISPR nuclease. As such, modification(s) at the 2’
Attorney Docket No.: 063586-537001WO position of either one or both of the 2nd and 3rd nucleotides at the 5’ end of the DR sequence would interfere with CRISPR nuclease/guide RNA interaction, thereby reducing gene editing efficiency. Based on the surprising discoveries reported herein, specific modification patterns of gRNAs have been designed and tested to identify those that enhance CRISPR nuclease activity and thus gene editing efficiency. Accordingly, provided herein are modified guide RNAs and gene editing systems comprising such and a Type V CRISPR nuclease that recognizes the modified RNA, as well as methods of using the gene editing system to implement genetic editing of target genes in host cells. I. CRISPR Nuclease-Containing Gene Editing Systems with Modified Guide RNAs The gene editing system provided herein comprises (a) a Type V CRISPR nuclease polypeptide or a nucleic acid (e.g., mRNA or a vector such as a viral vector) encoding the Type V CRISPR nuclease polypeptide, and (b) a modified guide RNA, which is recognizable by the Type V CRISPR nuclease polypeptide. 1. Type V CRISPR Nuclease Polypeptides CRISPR nuclease refers to an RNA-guided endonuclease in a CRISPR editing system that is capable of cleaving DNA at specific sites guided by an RNA guide. Type V CRISPR nuclease is a subclass of CRISPR nucleases. See, e.g., Tong et al., Front. Cell Dev. Biol., 2021, Vol.8, doi:10.3389/fcell.2020.622103. In some embodiments, the CRISPR nuclease used in the gene editing system disclosed herein comprises the amino acid sequence of SEQ ID NO: 1. See Table 1 below. In other embodiments, the CRISPR nuclease may comprise an amino acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. The “percent identity” (a.k.a., sequence identity) of two nucleic acids or of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol.215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength-12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST
Attorney Docket No.: 063586-537001WO protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res.25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. In some instances, the CRISPR nuclease of the present disclosure has enzymatic activity, e.g., nuclease or endonuclease activity similar to SEQ ID NO:1, and comprising an amino acid sequence, which differs from the amino acid sequences of SEQ ID NO: 1 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods. In some instances, the CRISPR nuclease may comprise one or more conservative amino acid substitutions as compared with SEQ ID NO: 1. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In some examples, the CRISPR nuclease comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or above) identical to SEQ ID NO:1 and one or more mutations relative to SEQ ID NO: 1. In some examples, the mutations may be at position P14, D32, I61, E311, T338, E736, or a combination thereof in SEQ ID NO:1. In some instances, the mutations are amino acid residue substitutions, for example, at the above noted position(s). In some examples, the amino acid residue substitution is P14R, D32R, I61R, E311R, T338G, E736G, or a combination thereof. In specific examples, the mutations are at positions P14R, D32R, I61R, and E311R in SEQ ID NO: 1, for example, P14R, D32R, I61R, and E311R. In other examples, the mutations are at positions P14R, D32R, I61R, E311, T338, and E736, for example, P14R, D32R, I61R, E311R, T338G, and E736G.
Attorney Docket No.: 063586-537001WO Table 1 below provided the amino acid sequences of exemplary CRISPR nuclease. Table 1. Exemplary CRISPR Nucleases Description Sequence SEQ ID NO
Attorney Docket No.: 063586-537001WO E736G) with EIHETLVDITNTHGENIVFTIKNDNLYIVFSYRSEFEKEEVNFAKTVGLDVN NLS and FKHAFFVGSEKDNCHLDGYINLYKYLLEHDEFTNLLTEDERKDYEELSKVVT peptide linkers FCPFENQLLFARYNKMSKFCKKEQVLSKLLYALQKKLKDENRTKEYIYVSCV
be found, e.g., in WO2021050534, WO2022192391, and WO2024020567, the relevant disclosures of each of which are incorporated by reference herein for the subject matter and purpose referenced herein. In some embodiments, the Type V CRISPR nuclease polypeptide provided herein may be a fusion polypeptide, which further comprises one or more functional elements. In some instances, the one or more functional elements are heterologous to the nuclease moiety in the CRISPR nuclease fusion polypeptide. As used herein, the terms “fusion” and “fused” refer to the joining of at least two nucleotide or protein molecules. For example, “fusion” and “fused” can refer to the joining of at least two polypeptide domains that are encoded by separate genes in nature. The fusion can be an N-terminal fusion, a C-terminal fusion, or an intramolecular fusion. In some aspects, the domains are transcribed and translated to produce a single polypeptide. In some examples, the one or more functional elements may comprise one or more nuclear localization signal(s) (NLSs), which may be located at either the N-terminus or the C- terminus. In specific examples, the fusion polypeptide may comprise a first NLS located at the N-terminus and a second NLS located at the C-terminus. The first and second NLS fragments may be identical. Alternatively, the two NLS fragments may be different. In some examples, the one or more functional elements may comprise one or more peptide linkers, which may be located between two functional domains in the fusion polypeptide, for example, between an NLS and the nuclease moiety. Exemplary NLS and peptide linker are provided in Table 1 above. In one example, the CRISPR nuclease polypeptide may comprise, from N-terminus to C-terminus, a first NLS, a first peptide linker, a nuclease moiety, a second peptide linker, and a second NLS. One specific example is SEQ ID NO: 6 provided in Table 1 above.
Attorney Docket No.: 063586-537001WO B. Modified RNA Guides The modified RNA guide provided herein comprises a spacer sequence (SP) and a direct repeat (DR) sequence. The DR sequence is recognizable by the Type V CRISPR nuclease disclosed herein. In some instances, the spacer sequence is located 3’ to the DR sequence. The modified RNA guide contains specific modification patterns at both 5’ and 3’ end as disclosed herein. In some instances, the modified RNA guide may contain one or more additional nucleotides at the 5’ end. In some embodiments, the modified RNA guide has a structure of 5’-N1-N2-DR-SP-3’ (Formula I), in which each of N1 and N2 independently is absent or a nucleotide; DR is a direct repeat sequence recognizable by a Type V CRISPR nuclease; and SP is a spacer sequence. In some examples, each of N1 and N2 is nucleotide. For example, N1 is G and N2 is A. In other examples, both N1 and N2 are absent. Such a modified RNA has a structure of 5’- DR-SP-3’. (i). Spacer Sequence The spacer sequence in the modified guide RNA is specific to a target sequence within a genomic site. The target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5’-NTTR-3’ (N being any of A, T, G, and C; and R being A or G), which is located 5’ to the target sequence. As used herein, the term “protospacer adjacent motif” or “PAM” refers to a DNA sequence adjacent to a target sequence to which a complex comprising an RNA guide (e.g., the modified RNA guide disclosed herein) and a CRISPR nuclease binds. In a double- stranded DNA molecule, the strand containing the PAM motif is called the “PAM-strand” and the complementary strand is called the “non-PAM strand.” The RNA guide binds to a site in the non-PAM strand that is complementary to a target sequence disclosed herein. As used herein, the term “spacer” or “spacer sequence” is a portion in an RNA guide that is the RNA equivalent of a target sequence (a DNA sequence) in the gene for editing. The spacer contains a sequence capable of binding to the non-PAM strand via base-pairing at the site complementary to the target sequence (in the PAM strand). Such a spacer is also known as specific to the target sequence. In some instances, the spacer may be at least 75% identical to the target sequence (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%), except for the RNA-DNA sequence difference. In some instances, the spacer may be 100% identical to the target sequence except for the RNA-DNA sequence difference.
Attorney Docket No.: 063586-537001WO As used herein, the term “target sequence” refers to a DNA fragment adjacent to a PAM motif (on the PAM strand). The complementary region of the target sequence is on the non-PAM strand. A target sequence may be immediately adjacent to the PAM motif. Alternatively, the target sequence and the PAM may be separately by a small sequence segment (e.g., up to 5 nucleotides, for example, up to 4, 3, 2, or 1 nucleotide). A target sequence may be located at the 3’ end of the PAM motif or at the 5’ end of the PAM motif, depending upon the CRISPR nuclease that recognizes the PAM motif, which is known in the art. For example, a target sequence is located at the 3’ end of a PAM motif for a CRISPR nuclease such as those disclosed herein. As used herein, the term “adjacent to” refers to a nucleotide or amino acid sequence in close proximity to another nucleotide or amino acid sequence. In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if no nucleotides separate the two sequences (i.e., immediately adjacent). In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if a small number of nucleotides separate the two sequences (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides). In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by up to 2 nucleotides, up to 5 nucleotides, up to 8 nucleotides, up to 10 nucleotides, up to 12 nucleotides, or up to 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by 2-5 nucleotides, 4-6 nucleotides, 4-8 nucleotides, 4-10 nucleotides, 6-8 nucleotides, 6-10 nucleotides, 6-12 nucleotides, 8-10 nucleotides, 8-12 nucleotides, 10-12 nucleotides, 10-15 nucleotides, or 12-15 nucleotides. In some embodiments, the spacer sequence of the RNA guide provided herein may have a length of between 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and is complementary to a non-PAM strand sequence. In some examples, the spacer sequence has a length of 15-25 nucleotides, e.g., 16-20 nucleotides. In some embodiments, the spacer sequence is designed to be complementary to a specific DNA strand, e.g., of a genomic locus. In some examples, the spacer sequence has 16-nucleotide in length. In other examples, the spacer sequence has 18-nucleotide in length. In yet other examples, the spacer sequence has 20-nucleotide in length. It is reported that gRNAs for the CRISPR nucleases disclosed herein that have a shortened spacer sequence (e.g., 16-nt) exhibit higher gene
Attorney Docket No.: 063586-537001WO editing efficiencies relative to the counterpart gRNAs having a longer spacer sequence, regardless of the target gene intended for editing. In some instances, the shortened spacer sequence may have 3’ end truncations relative to the counterpart spacer sequence that matches the target sequence (i.e., having the same length as the target sequence, which may be 20-nt in length). The 3’ end truncation may include a deletion of up to 5 nucleotides, for example, up to 4 nucleotides, up to 3 nucleotides, or up to 2 nucleotides. In some instances, the spacer sequence may be 18-nt in length when the cognate target sequence is 20-nt in length. In other instances, the spacer sequence may be 17-nt in length when the cognate target sequence is 20-nt in length. In yet other instances, the spacer sequence may be 16-nt in length when the cognate target sequence is 20-nt in length. As used herein, the term “complementary” refers to a first polynucleotide (e.g., a spacer sequence of an RNA guide) that has a certain level of complementarity to a second polynucleotide (e.g., the complementary sequence of a target sequence) such that the first and second polynucleotides can form a double-stranded complex via base-pairing to permit an effector polypeptide that is complexed with the first polynucleotide to act on (e.g., cleave) the second polynucleotide. In some embodiments, the first polynucleotide may be substantially complementary to the second polynucleotide, i.e., having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the second polynucleotide. In some embodiments, the first polynucleotide is completely complementary to the second polynucleotide, i.e., having 100% complementarity to the second polynucleotide. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. In some embodiments, the PAM strand is a coding (e.g., sense) strand. In other embodiments, the PAM strand is a non-coding (e.g., antisense strand). Since an RNA guide binds the non-PAM strand via base-pairing, the non-PAM strand is also known as the target strand, while the PAM strand is also known as the non-target strand. In some embodiments, a spacer sequence provided herein may comprise only RNA bases. In some embodiments, the spacer sequence may comprise a DNA base (e.g., the spacer comprises at least one thymine). In some embodiments, the spacer sequence may comprise RNA bases and DNA bases (e.g., the spacer sequence comprises at least one thymine and at least one uracil).
Attorney Docket No.: 063586-537001WO (ii). Direct Repeat Sequence The direct repeat (DR) sequence in the modified guide RNA disclosed herein can be recognized by the Type V CRISPR nuclease polypeptide disclosed herein. In some embodiments, the direct repeat sequence of the RNA guide has a length of between 12-100, 13-75, 14-50, or 15-40 nucleotides (e.g., 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, or 40 nucleotides). In some embodiments, the direct repeat sequence is a sequence provided in Table 2 or a portion thereof. Table 2. CRISPR Nuclease Direct Repeat Sequences SEQ ID NO Direct Repeat Sequence 7 CUUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAAC
In some instances, the direct repeat sequence is any one of SEQ ID NOs: 7-16 provided in Table 2 above. In one example, the direct repeat sequence comprises (e.g., consists of) SEQ ID NO: 7. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 2. In some examples, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to SEQ ID NO: 7. Additional direct repeat sequence for use in constructing the RNA guides provided herein can be found, e.g., in WO2021050534 or WO2022192391, the relevant disclosures of each of which are incorporated by reference herein for the subject matter and purpose referenced herein. (iii) Modification of RNA Guides The guide RNAs provided herein contain modifications, including 2’-O-methylation and/or phosphorothioate bond modifications, which may be located at the 5’ end, the 3’ end,
Attorney Docket No.: 063586-537001WO or both of the guide RNA. For example, 1, 2, or 3 nucleotides at the 5’ end of the guide RNA may contain 2’-O-methylation, phosphorothioate bond modification, or a combination thereof. Alternatively or in addition, 1, 2, or 3 nucleotides at the 3’ end of the guide RNA may contain 2’-O-methylation, phosphorothioate bond modification, or a combination thereof. It is reported herein that modifications at the 2’ position of the 2nd and 3rd nucleotides at the 5’ end of the DR sequence may interfere with binding of the CRISPR nuclease to the guide RNA. Accordingly, the 2’ position of the 2nd and/or 3rd nucleotides at the 5’ end of the DR sequence may not be modified. In other words, the 2’ position of the 2nd nucleotide, the 2’ position of the 3rd nucleotide, or both at the 5’ end of the DR sequence is an -OH group. Exemplary modification patterns are provided in Table 5 below. In one example, the modification pattern is mod2. In another example, the modification pattern is mod4. In yet another example, the modification pattern is mod6. In still another example, the modification pattern is mod7. The present disclosure shows that gRNAs for the CRISPR nucleases disclosed herein that have such modification patterns would exhibit higher gene editing efficiencies relative to the unmodified counterparts or gRNAs having the same sequence but different modifications, regardless of the target gene intended for editing. (iv) Modification of Nucleic Acids A nucleic acid component in the gene editing system provided herein (e.g., an mRNA molecule encoding the Type V CRISPR nuclease polypeptide disclosed herein) may include one or more modifications. Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone), and any combination thereof. Some of the exemplary modifications provided herein are described in detail below. The nucleic acid such as the encoding mRNA may include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). One or more atoms of a purine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside
Attorney Docket No.: 063586-537001WO linkage. In some embodiments, the gRNA or any of the nucleic acid sequences encoding components of the composition may comprise an abasic site (i.e., a location that does not have a purine or a pyrimidine). Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein. In some embodiments, the modification may include a chemical or cellular induced modification. For example, some nonlimiting examples of intracellular RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to guide RNA-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210. Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (e.g., backbone structures) may exist at various positions in the sequence. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the sequence, such that the function of the sequence is not substantially decreased. The sequence may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). In some embodiments, sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar at one or more ribonucleotides of the sequence may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of a sequence include, but are not limited to, sequences including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages. Sequences having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art,
Attorney Docket No.: 063586-537001WO modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, a sequence will include ribonucleotides with a phosphorus atom in its internucleoside backbone. Modified sequence backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3’-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3’-5’ linkages, 2’-5’ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’. Various salts, mixed salts and free acid forms are also included. In some embodiments, the sequence may be negatively or positively charged. The modified nucleotides, which may be incorporated into the sequence, can be modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates). The α-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5’-O-(1-thiophosphate)-adenosine, 5’-O-(1-thiophosphate)-cytidine (a-thio-cytidine), 5’-O-(1-thiophosphate)-guanosine, 5’-O-(1-thiophosphate)-uridine, or 5’-O-(1- thiophosphate)-pseudouridine).
Attorney Docket No.: 063586-537001WO Other internucleoside linkages that may be employed according to the present invention, including internucleoside linkages which do not contain a phosphorous atom, are described herein. In some embodiments, the sequence may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into sequence, such as bifunctional modification. Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4’-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-beta-D-arabino- pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione), troxacitabine, tezacitabine, 2’-deoxy-2’-methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-1- beta-D-arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4- palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5’-elaidic acid ester). In some embodiments, the sequence includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197) In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5- aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl- pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl- pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2- thio-pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside
Attorney Docket No.: 063586-537001WO selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1- methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza- zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1- methyl-pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2, 6-diaminopurine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2- aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In some embodiments, mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza- guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7- deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6- methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8- oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio- guanosine, and N2,N2-dimethyl-6-thio-guanosine. The sequence may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally-occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the sequence, or in a given predetermined sequence region thereof. In some embodiments, the sequence includes a pseudouridine. In some embodiments, the sequence includes an inosine, which may aid in the immune system characterizing the sequence as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability/reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res.25, 1283-1284, which is incorporated by reference in its entirety. In some embodiments, any RNA sequence described herein may comprise an end modification (e.g., a 5’ end modification or a 3’ end modification). In some embodiments, the
Attorney Docket No.: 063586-537001WO end modification is a chemical modification. In some embodiments, the end modification is a structural modification. See disclosures herein. C. Exemplary Gene Editing Systems In some instances, the gene editing system provided herein may comprise the Type V CRISPR nuclease polypeptide, which can be prepared by a conventional approach or as disclosed herein, and the modified guide RNA. In some instances, the Type V CRISPR nuclease polypeptide and the modified RNA may form a ribonucleoprotein (RNP) complex. In other instances, the gene editing system provided herein may comprise a nucleic acid encoding the Type V CRISPR nuclease polypeptide and the modified guide RNA. In some examples, the nucleic acid can be an mRNA molecule encoding the Type V CRISPR nuclease polypeptide. Alternatively, the nucleic acid can be a vector, such as a viral vector (e.g., a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector). The gene editing systems provided herein may further comprise one or more lipid excipient, which may form lipid nanoparticles (LNP). One or more of the components of the gene editing system (e.g., the encoding mRNA) may be associated with the lipid excipients to facilitate delivery of the gene editing system components to host cells. Exemplary LNP formulations are provided in the Examples below. II. Preparation of Gene Editing System Components The present disclosure provides methods for production of components of the gene editing systems disclosed herein, e.g., the modified RNA guide and the CRISPR nuclease, as well as methods for complexing the modified RNA guide and the CRISPR nuclease. A. RNA Guide In some embodiments, the modified RNA guide disclosed herein can be made by chemical synthesis, during which the desired modifications can be incorporated. Methods for synthesize modified RNA molecules are known in the art. In some embodiments, the RNA guide is made using chemical synthetic methods. In some embodiments, the RNA guide is synthesized using one or more modified nucleotide, e.g., as described above. B. CRISPR nuclease In some embodiments, the Type V CRISPR nuclease polypeptide disclosed in the
Attorney Docket No.: 063586-537001WO present disclosure can be prepared by the conventional recombinant technology. For example, nucleic acids encoding the Type V CRISPR nuclease polypeptide can be cloned into one expression vector, the coding sequence being in operable linkage to a suitable promoter. The expression vector can be introduced into suitable host cells, which can be cultured under suitable conditions allowing for the production of the polypeptide, which can be isolated and purified from the cell culture. Optionally, the Type V CRISPR nuclease polypeptide can be complexed with an RNA guide (e.g., the modified guide RNA provided herein) to form a complex. Suitable host cells are not particularly limited, and various known cells can be preferably used. Specific examples of the host cell include bacteria such as E. coli, yeasts (budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, and animal cells (for example, CHO cells, COS cells and HEK293 cells). The method for transferring the expression vector described above into host cells, i.e., the transformation method, is not particularly limited, and known methods such as electroporation, the calcium phosphate method, the liposome method and the DEAE dextran method can be used. After a host is transformed with the expression vector, the host cells may be cultured, cultivated or bred, for production of the Type V CRISPR nuclease polypeptide. After expression of the Type V CRISPR nuclease polypeptide, the host cells can be collected and Type V CRISPR nuclease polypeptide purified from the cultures etc. according to conventional methods (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, etc.). A variety of methods can be used to determine the level of production of the Type V CRISPR nuclease polypeptide in a host cell. Such methods include, but are not limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the Type V CRISPR nuclease polypeptide or a labeling tag as described elsewhere herein. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (MA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See, e.g., Maddox et al., J. Exp. Med.158:1211 [1983]). The present disclosure provides methods of in vivo expression of the Type V CRISPR nuclease polypeptide in a cell, comprising providing a polyribonucleotide encoding the Type V CRISPR nuclease polypeptide to a host cell wherein the polyribonucleotide encodes the Type V CRISPR nuclease polypeptide, expressing the CRISPR nuclease in the cell, and
Attorney Docket No.: 063586-537001WO obtaining the Type V CRISPR nuclease polypeptide from the cell. The present disclosure further provides methods of in vivo expression of the Type V CRISPR nuclease polypeptide in a cell, comprising providing a polyribonucleotide (e.g., an mRNA molecule) encoding the CRISPR nuclease to a host cell located in a subject such as a human patient. In some examples, the polyribonucleotide encoding the Type V CRISPR nuclease polypeptide can be delivered to the cell with an RNA guide and, once expressed in the cell, the Type V CRISPR nuclease polypeptide and the RNA guide form a complex. C. CRISPR Nuclease-gRNA Complexes In some embodiments, a modified guide RNA as disclosed herein can be complexed with the Type V CRISPR nuclease polypeptide also disclosed herein to form a ribonucleoprotein (RNP) complex. In some embodiments, complexation of the modified RNA guide and the CRISPR nuclease occurs at a temperature lower than about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, or 55°C. In some embodiments, the RNA guide does not dissociate from the CRISPR nuclease at about 37°C over an incubation period of at least about any one of 10 mins, 15 mins, 20 mins, 25 mins, 30 mins, 35 mins, 40 mins, 45 mins, 50 mins, 55 mins, 1 hr, 2 hrs, 3 hrs, 4 hrs, or more hours. In some embodiments, the modified RNA guide and the Type V CRISPR nuclease polypeptide are complexed in a complexation buffer. In some embodiments, the Type V CRISPR nuclease polypeptide is stored in a buffer that is replaced with a complexation buffer to form a complex with the RNA guide. In some embodiments, the Type V CRISPR nuclease polypeptide is stored in a complexation buffer. In some embodiments, the complexation buffer has a pH in a range of about 7.3 to 8.6. In one embodiment, the pH of the complexation buffer is about 7.3. In one embodiment, the pH of the complexation buffer is about 7.4. In one embodiment, the pH of the complexation buffer is about 7.5. In one embodiment, the pH of the complexation buffer is about 7.6. In one embodiment, the pH of the complexation buffer is about 7.7. In one embodiment, the pH of the complexation buffer is about 7.8. In one embodiment, the pH of the complexation buffer is about 7.9. In one embodiment, the pH of the complexation buffer is about 8.0. In one embodiment, the pH of the complexation buffer is about 8.1. In one embodiment, the pH of the complexation buffer is about 8.2. In one embodiment, the pH of the complexation buffer is about 8.3. In one embodiment, the pH of the complexation buffer
Attorney Docket No.: 063586-537001WO is about 8.4. In one embodiment, the pH of the complexation buffer is about 8.5. In one embodiment, the pH of the complexation buffer is about 8.6. In some embodiments, the Type V CRISPR nuclease polypeptide can be overexpressed and complexed with the RNA guide in a host cell prior to purification as described herein. In other embodiments, mRNA or DNA encoding the Type V CRISPR nuclease polypeptide can be introduced into a cell so that the Type V CRISPR nuclease polypeptide is expressed in the cell. In some embodiments, the RNA guide is also introduced into the cell, whether simultaneously, separately, or sequentially from a single mRNA or DNA construct, such that the ribonucleoprotein complex is formed in the cell. In some instances, the cell can be a target cell (e.g., a liver cell). III. Gene Editing Methods The disclosure also provides methods of genetically editing a target gene in host cells such as liver cells using any of the gene editing systems disclosed herein. In some instances, the methods comprise introducing any of the gene editing systems disclosed herein into cultured target cells (in vitro editing). For example, a modified RNA guide and a Type V CRISPR nuclease polypeptide as provided herein can be introduced into a population of cultured cells. Alternatively, the methods comprise introducing the gene editing system into a subject such as a human patient to achieve editing of a target gene in vivo. The modified RNA guide and the Type V CRISPR nuclease polypeptide can form a ribonucleoprotein complex, which can be introduced into target cells. Alternatively, the modified RNA guide and the Type V CRISPR nuclease polypeptide can be encoded by a nucleic acid vector(s), which can be introduced into the target cells. In another example, an mRNA molecule encoding the Type V CRISPR nuclease polypeptide can be introduced into target cells. Alternatively or in addition, the RNA guide can be introduced directly into the target cells. Any of the gene editing systems disclosed herein may be used to genetically engineered a target gene in target cells of interest (e.g., liver cells). A. Delivery of Gene Editing System Components of any of the gene editing systems disclosed herein may be formulated, for example, including a carrier, such as a carrier and/or a polymeric carrier, e.g., a liposome, and delivered by known methods to target cells (e.g., a prokaryotic, eukaryotic, plant,
Attorney Docket No.: 063586-537001WO mammalian, etc.). In some examples, the target cells are liver cells. Such methods include, but not limited to, transfection (e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV)), microinjection, microprojectile bombardment (“gene gun”), fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome- mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof. In some embodiments, the method comprises delivering one or more nucleic acids (e.g., nucleic acids encoding the Type V CRISPR nuclease polypeptide disclosed herein, and a modified guide RNA as also disclosed herein), one or more transcripts thereof, and/or a pre- formed RNA guide/CRISPR nuclease complex to target cells. In some instances, a modified RNA guide and an RNA (e.g., mRNA) encoding the Type V CRISPR nuclease polypeptide are delivered together in a single composition. Alternatively, a modified RNA guide and an RNA (e.g., mRNA) encoding the Type V CRISPR nuclease polypeptide are delivered in separate compositions. For example, the modified RNA guide and the RNA (e.g., mRNA) encoding the Type V CRISPR nuclease polypeptide delivered in separate compositions are delivered using the same delivery technology. In other examples, the modified RNA guide and an RNA (e.g., mRNA) encoding the Type V CRISPR nuclease polypeptide delivered in separate compositions are delivered using different delivery technologies. Exemplary intracellular delivery methods, include, but are not limited to: viruses, such as AAV, or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, lipid nanoparticles, or cationic polymers (e.g., DEAE-dextran or polyethylenimine); non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons; particle-based methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection. In some embodiments, a lipid nanoparticle comprises an mRNA encoding the Type V CRISPR nuclease polypeptide (e.g., those provided herein such as the one in Table 1 above), an RNA guide, or an mRNA encoding the CRISPR nuclease polypeptide and the RNA guide can be used for delivering the gene editing components to target cells such as to liver cells. Any suitable LNP components that have been used for delivering nucleic acids to cells can also be used in the instant disclosure, for example, the LNP particles used for delivering the COVID-19 mRNA vaccines as reported in Schoenmaker et al., International J.
Attorney Docket No.: 063586-537001WO Pharmaceutics, 601 (2021) 120586, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. Effectiveness of target gene editing in the target cells can be examined via conventional technology and/or those provided in Examples below. B. Genetically Modified Cells Any of the gene editing systems disclosed herein can be delivered to a variety of cells to genetically edit a target gene, which can lead to substantially reduced or no expression of the target gene. In some embodiments, the target cells can be cultured cells or a co-culture of two or more types of cells. In some examples, the target cells can be obtained from a living organism (e.g., a mammal such as a human subject) and maintained in a cell culture. In some embodiments, the cell can be a single-cellular organism. For example, the cell can be a prokaryotic cell. In some examples, the cell is a bacterial cell or derived from a bacterial cell. In other examples, the cell is an archaeal cell or derived from an archaeal cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a plant cell or derived from a plant cell. In some embodiments, the cell is a fungal cell or derived from a fungal cell. In some embodiments, the cell is an animal cell or derived from an animal cell. In some embodiments, the cell is an invertebrate cell or derived from an invertebrate cell. In some embodiments, the cell is a vertebrate cell or derived from a vertebrate cell. In some embodiments, the cell is a mammalian cell or derived from a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a zebra fish cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is derived from a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, CHO, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, the cell is an immortal or immortalized cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a stem cell such as a totipotent stem cell (e.g., omnipotent), a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell, or an unipotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or derived from an iPSC. In some embodiments, the cell is a differentiated cell. For example, in some embodiments, the differentiated cell is a liver cell (e.g., a hepatocyte), a biliary cell (e.g., a cholangiocyte), a
Attorney Docket No.: 063586-537001WO stellate cell, a Kupffer cell, a liver sinusoidal endothelial cell, a muscle cell (e.g., a myocyte), a fat cell (e.g., an adipocyte), a bone cell (e.g., an osteoblast, osteocyte, osteoclast), a blood cell (e.g., a monocyte, a lymphocyte, a neutrophil, an eosinophil, a basophil, a macrophage, a erythrocyte, or a platelet), a nerve cell (e.g., a neuron), an epithelial cell, an immune cell (e.g., a lymphocyte, a neutrophil, a monocyte, or a macrophage), a fibroblast, or a sex cell. In some embodiments, the cell is a terminally differentiated cell. For example, in some embodiments, the terminally differentiated cell is a neuronal cell, an adipocyte, a cardiomyocyte, a skeletal muscle cell, an epidermal cell, or a gut cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a Natural Killer (NK) cell. In some embodiments, the immune cell is a Tumor Infiltrating Lymphocyte (TIL). In some embodiments, the cell is a mammalian cell, e.g., a human cell or a murine cell. In some embodiments, the murine cell is derived from a wild-type mouse, an immunosuppressed mouse, or a disease-specific mouse model. In some embodiments, the cell is a cell within a living tissue, organ, or organism. Any of the gene editing systems or modified cells (e.g., modified liver cells) generated using such a gene editing system as disclosed herein may be used for treating a disease associated with the target gene. In some embodiments, provided herein is a method for treating a disease associated with a target gene using any of the gene editing systems disclosed herein, which comprise a modified guide RNA specific to the target gene. The method may comprise administering to a subject (e.g., a human patient) in need of the treatment any of the gene editing systems disclosed herein. The gene editing system may be delivered to a specific tissue (e.g., liver) or specific type of cells (e.g., liver cells) where the gene edit is needed. The gene editing system may comprise LNPs encompassing one or more of the components, one or more vectors (e.g., viral vectors) encoding one or more of the components, or a combination thereof. Components of the gene editing system may be formulated to form a pharmaceutical composition, which may further comprise one or more pharmaceutically acceptable carriers. In some embodiments, provided herein is a composition comprising the gene editing system disclosed herein or components thereof. Such a composition can be a pharmaceutical composition. A pharmaceutical composition that is useful may be prepared, packaged, or sold in a formulation for a suitable delivery route, e.g., parenteral, intra-lesional, intra-organ or another route of administration. A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit
Attorney Docket No.: 063586-537001WO doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition (e.g., the gene editing system or components thereof), which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. A formulation of a pharmaceutical composition suitable for parenteral administration may comprise the active agent (e.g., the gene editing system or components thereof or the modified cells) combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such a formulation may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Some injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Some formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Some formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the cells, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulation may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or saline. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which that are useful include those which may comprise the cells in a packaged form, in a liposomal preparation, or as a component of a biodegradable polymer system. Some compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. IV. Kits for Genetic Editing of Target Genes The present disclosure also provides kits that can be used, for example, to carry out a gene editing method described herein for genetical editing of a target gene as disclosed herein. In some embodiments, the kits include a modified guide RNA as disclosed herein and a Type V CRISPR nuclease polypeptide as also disclosed herein (e.g., those provided herein
Attorney Docket No.: 063586-537001WO such as the one provided in Table 1). In some embodiments, the kits include the modified RNA guide and the Type V CRISPR nuclease polypeptide. In other embodiments, the kits include a polynucleotide that encodes the CRISPR nuclease polypeptide. In some examples, the polynucleotide is comprised within a vector, e.g., as described herein. The CRISPR nuclease polypeptide (or polynucleotide encoding the CRISPR nuclease) and the modified RNA guide (e.g., as a ribonucleoprotein) can be packaged within the same or other vessel within a kit or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use. The Type V CRISPR nuclease polypeptide and the modified RNA guide can be packaged within the same or other vessel within a kit or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use. The kits can additionally include, optionally, a buffer and/or instructions for use of the modified RNA guide and the Type V CRISPR nuclease polypeptide. General techniques The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed.1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed.1987); Introuction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E.
Attorney Docket No.: 063586-537001WO Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed.1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (lRL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.). Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. EXAMPLE 1: CRISPR Nuclease-Mediated Editing of Target Sequences in Primary Human Hepatocyte (PHH) Donor Cells Using RNA Guides with End Modifications This Example describes genomic editing of the PCSK9 gene using a variant CRISPR nuclease (SEQ ID NO: 3) introduced into primary human hepatocytes (PHH) by mRNA or purified protein with synthetic RNA guides containing conventional modifications (2’-O methylation and phosphorothioate (PS) bond modifications at both 5’ and 3’ ends). On-target indels were determined and compared between cells transfected with (a) nuclease and unmodified RNA guides and (b) nuclease and RNA guides modified at both ends. Structural information of those RNA guides can be found in Table 3 below. Table 3. Target and RNA Guide Sequences for PCSK9 and HAO1 Guide Mod PAM Target DR Sequence Spacer Sequence RNA Guide Sequence Se uence ǂ U U G C A A G G Q
Attorney Docket No.: 063586-537001WO Guide Mod PAM Target DR Sequence Spacer Sequence RNA Guide Sequence Sequence ǂ none CUUGUUGUAUAUGU U CA A A G CC ID U U A G A A U A U U A U A A G G Q
The modification patterns of the gRNAs are summarized in Table 4 below: Table 4. RNA Guide Modification Patterns Modification name Modification information Generic guide sequence SEQ NO
Attorney Docket No.: 063586-537001WO Both ends three PS bonds and three 2’- mC*mU*mU*GUUGUAUAUGU 34 O-methyl bases on 5’ end CCUUUUAUAGGUAUUAAAC AACNNNNNNNNNNNNNNNN
Electroporation of primary human hepatocyte (PHH) donor cells with mRNA+gRNA A CRISPR nuclease polypeptide of SEQ ID NO: 6, comprising a nuclear localization signal (NLS; SEQ ID NO: 4) at both the N-terminus and C-terminus, which is connected to the nuclease variant of SEQ ID NO: 3 via a flexible peptide linker (SEQ ID NO: 5), was cloned into an in vitro transcription (IVT) backbone and reverse transcribed into mRNA using an in vitro transcription kit. The full amino acid sequence of the CRISPR nuclease polypeptide and the sequences of the components thereof are provided in Table 1 above. A working solution of mRNA for expression was prepared in water at a concentration of 1 mg/mL. PHH cells from human donors (from ThermoFisher or Lonza) were thawed from liquid nitrogen quickly in a 37oC water bath. The cells were added to pre-warmed hepatocyte recovery media (ThermoFisher, CM7000) and centrifuged for 10 minutes. The cell pellet was resuspended in appropriate volume of hepatocyte plating Medium (Williams’ Medium E, ThermoFisher A1217601 supplemented with Hepatocyte Plating Supplement Pack (serum- containing), ThermoFisher CM3000). The cells were subjected to a trypan blue viability count using the Vi-CELL BLU cell counter. The desired number of viable cells were then washed in PBS and resuspended in P3 buffer + supplement (Lonza, V4SP-3096) and transfection enhancer oligo. Resuspended cells were dispensed at 125,000 cells/reaction into Lonza 96- well electroporation plates. The mRNA encoding the nuclease was mixed with synthetic RNA guides (Table 3) at a 1:1 volume ratio. mRNA/guide RNA mixtures were added to each reaction at a final mRNA concentration of 25 nM, 6.25 nM, 1.56 nM, or 0.39 nM. The plates were electroporated using an electroporation device (program DS-150, Lonza 4D-nucleofector).5 minutes following electroporation, pre-warmed Hepatocyte plating medium was added to each well and mixed very gently by pipetting. For each technical replicate plate, 125,000 cells of diluted nucleofected cells were plated into a pre-warmed collagen-coated 96-well plate (ThermoFisher) with wells containing Hepatocyte plating
Attorney Docket No.: 063586-537001WO medium. The cells were then incubated in a 37oC incubator. The media was changed to hepatocyte maintenance media (Williams’ Medium E, ThermoFisher A1217601 supplemented with William’s E medium Cell Maintenance Cocktail, ThermoFisher CM 4000) after the cells attached after 4 hours. After 3 days post electroporation, cells in the wells were harvested using Accutase and transferred to 96-well twin.tec® PCR plates (Eppendorf) and centrifuged. Media was flicked off and cells were resuspended in DNA extraction buffer (QuickExtractTM). Samples were then cycled in a PCR machine at 65°C for 15 min, 68°C for 15 min, 98°C for 10 min. Samples were then frozen at -20°C, and subsequently analyzed for PCSK9 or HAO1 indels using Next Generation Sequencing (NGS), as described below. Electroporation of primary human hepatocyte (PHH) donor cells with RNPs To test the same gRNA modifications when delivered to PHH cells as RNPs, the mRNA expressing the CRISPR nuclease polypeptide of SEQ ID NO: 6 described above was expressed and purified. Briefly, the CRISPR nuclease polypeptide was recombinantly expressed with a N-terminal 6xHis affinity tag followed by a SUMO domain for solubility. The CRISPR nuclease polypeptide was purified using Ni-NTA affinity and heparin chromatography, and the SUMO tag was then removed by treatment with commercially- obtained Ulp1 SUMO protease. Cleaved tag was removed by subsequent Ni-NTA affinity chromatography and the CRISPR nuclease polypeptide was finally subjected to size-exclusion chromatography as a final polishing step. RNP complexation reactions were made by mixing purified CRISPR nuclease (SEQ ID NO: 3) with a modified or unmodified RNA guide (Table A)) at a 1:1 (CRISPR nuclease:RNA guide) volume ratio (2.5:1 RNA guide:CRISPR nuclease molar ratio). Complexations were incubated on ice for 45-60 min. RNP mixtures were added to reaction at a final protein concentration of 5 µM, 0.625 µM, 0.156 µM, or 0.039 µM. PHH cells were electroporated as described above, incubated for 3 days, and harvested as described above. Samples were then frozen at -20°C, and subsequently analyzed for PCSK9 or HAO1 indels using Next Generation Sequencing (NGS), as described below. NGS library preparation and analysis Samples for Next Generation Sequencing (NGS) were prepared by rounds of PCR. The first round (PCR I) was used to amplify the genomic regions flanking the target site and add NGS adapters. The second round (PCR II) was used to add NGS indexes. Reactions were
Attorney Docket No.: 063586-537001WO then pooled, purified by column purification, and quantified on a fluorometer (Qubit). Sequencing runs were done using a 150 cycle NGS instrument (NextSeq v2.5) mid or high output kit and run on an NGS instrument (NextSeq 550). For NGS analysis, the indel mapping function used a sample’s fastq file, the amplicon reference sequence, and the forward primer sequence. For each read, a kmer-scanning algorithm was used to calculate the edit operations (match, mismatch, insertion, deletion) between the read and the reference sequence. In order to remove small amounts of primer dimer present in some samples, the first 30 nt of each read was required to match the reference and reads where over half of the mapping nucleotides are mismatches were filtered out as well. Up to 50,000 reads passing those filters were used for analysis, and reads were counted as an indel read if they contained an insertion or deletion. The % indels was calculated as the number of indel-containing reads divided by the number of reads analyzed (reads passing filters up to 50,000). The QC standard for the minimum number of reads passing filters was 10,000. Results from this Example show that the unmodified guides showed high activity in the RNP format and reduced activity with the mRNA+gRNA setting, suggesting the need for modifications in the context of mRNA delivery. The gRNAs with modifications at both ends (“bothends modifications” or “both end modification”) showed significantly worse activity in both the RNP and mRNA setting, indicating that certain modifications at either end or both ends of the gRNA may have negative impact on gene editing efficiency. EXAMPLE 2: gRNAs with Various End Modifications Designed Based on Nuclease Structure for Improving Nuclease Activity This example describes determination of the predicted structure of the CRISPR nuclease of SEQ ID NO: 3 when complexed with a gRNA and design of proper end modification patterns for gRNAs to improve nuclease activity. Structure of CRISPR Nuclease Polypeptide The CRISPR nuclease polypeptide of SEQ ID NO: 6, comprising the nuclease moiety of SEQ ID NO: 3, NLSs and peptide linkers as detailed in Example 1 above, was prepared following the method described in Example 1 above. The CRISPR nuclease polypeptide was complexed with synthetically-produced RNA guide and dsDNA oligos complementary to the spacer and PAM preference of the nuclease of SEQ ID NO: 3. Prior to structure determination, RNA was selected with a spacer 16 nt in length based
Attorney Docket No.: 063586-537001WO on improved thermal stability compared with a 20 nt-spacer guide RNA. The ternary complex was isolated by size-exclusion chromatography and applied to QuantiFoil Cu 300 R2/1 grid at 0.5 mg/mL using a Vitrobot Mark IV (ThermoFisher). Micrographs were collected on a Titan Krios G4 TEM (ThermoFisher) with a Falcon 4i detector and 300 keV source using a 20 stage tilt and processed using cryoSPARC. Final map resolution is 3.22 angstroms (determined using 0.143 “gold-standard” cutoff) The atomic model was built using a manually-rebuilt Alphafold input structure and refined in Refmac5. Structural analysis of the CRISPR nuclease polypeptide complexed with an RNA guide demonstrated that nucleotides 2 and 3 of the 5’ direct repeat (DR) region engage in hydrogen bonds with the nuclease, specifically at the 2’ hydroxyl groups within those two nucleotides. FIG.2A. The close proximity of these contacts and the constraints on this form of intermolecular interaction suggests that methylation at those nucleotides in the DR would interfere with nuclease:RNA guide binding and result in reduced indel activity. In sum, consistent with the results reported in Example 1, structural analysis of the CRISPR nuclease polypeptide/gRNA complex suggests that 2’-O-methyl modifications on nucleotides 2 and 3 of the DR may interfere with nuclease binding to the RNA guide. Design of Modification Patterns for Guide RNAs Based on Nuclease Structural Analysis To assess the effect of various guide modifications on the 5’ end of the RNA guide, several different modification configurations were designed based on the nuclease structural analysis. The modification patterns are summarized in Table 5 below. Table 5. RNA Guide Modification Patterns Modification Modification information Generic guide sequence (N = SEQ Pattern base) ID NO
Attorney Docket No.: 063586-537001WO mod4 one PS bond on 5’ end C*UUGUUGUAUAUGUCCUU 38 UUAUAGGUAUUAAACAACN th PS b d d th 2’ O N* N* N* N
m = -O-met y mod ed base, w ere = , G, C, or U The spacer region represented by the “N” residues can be 16-20-nt in length, for example, 16-nt or 18-nt. Guides designated as “mod5” or “mod6” also contained additional modified nucleotides added to the 5’ end of the guide sequence (see Table 5 above). RNA guides targeting PCSK9 and HAO1 were designed with the various chemical modifications summarized in Table 5 above and ordered from Integrated DNA Technologies (IDT). The sequence and modification information of these RNA guides are provided in Table 6 below. The PAM, target sequence, DR sequence, and spacer sequence for the listed gRNAs are provided in Table 3 above. The unmodified and bothends modified counterparts for each of the guide RNAs are provide in Table 4 above. Table 6. Target and RNA Guide Sequences for PCSK9 and HAO1 Guides Mod RNA Guide Sequence A A ID
Attorney Docket No.: 063586-537001WO Guides Mod RNA Guide Sequence mod3 mC*UUGUUGUAUAUGUCCUUUUAUAGGUAUUAAA ID C A C UU A A D A D C A G UU A A ID A ID C A G UU A
Attorney Docket No.: 063586-537001WO Guides Mod RNA Guide Sequence mod2 C*U*U*GUUGUAUAUGUCCUUUUAUAGGUAUUAAA ID A ID C D A U UU
p p . . Editing Efficiency of Modified Guide RNAs The gene editing efficiencies of the modified guide RNAs targeting PCSK9 or HAO1 in PHH cells, coupled with the CRISPR nuclease polypeptide of SEQ ID NO: 6, were analyzed following the methods described in Example 1 above. FIG.2B shows PCSK9 and HAO1 indels in a PHH donor following electroporation of the mRNA encoding the CRISPR nuclease polypeptide of SEQ ID NO: 6 and the selected modified RNA guides. Mod2, mod4, and mod6 show significantly higher on-target indel activity across the three doses and four targets compared to unmod, mod1, mod3, and mod5. FIG.2C shows PCSK9 and HAO1 indels in a PHH donor following electroporation of the RNP comprising the CRISPR nuclease polypeptide of SEQ ID NO: 6 and the selected modified RNA guides. Unmod, mod4, and mod6 show significantly higher on-target indel activity across the three doses and four targets compared to mod1, mod2, mod3, and mod5. Mod2 showed higher activity than mod1, mod3, and mod5 in most targets, but not as high as unmod, mod4, or mod6. FIG.2D shows PCSK9 and HAO1 indels in a PHH donor following electroporation of the mRNA encoding the CRISPR nuclease polypeptide of SEQ ID NO: 6 and the selected modified RNA guides. All three modifications (mod2, mod4, mod6) demonstrated high on- target activity for guides tested, with unmodified guides demonstrating reduced activity when delivered with an mRNA expressing the nuclease. Specifically, mod4 showed the highest activity across the guides tested, while mod2 and mod6 were slightly lower for specific
Attorney Docket No.: 063586-537001WO guides compared to mod4. FIG.2E shows PCSK9 and HAO1 indels in a PHH donor following electroporation of the RNP comprising the CRISPR nuclease polypeptide of SEQ ID NO: 6 and the selected modified RNA guides. All three modifications (mod2, mod4, mod6) demonstrated high on- target activity for guides tested, with mod2 showing reduced activity compared to mod4 and mod6. Unlike the conditions where the nuclease was delivered as an mRNA, the unmodified guide showed higher indels compared to the modified guides in the RNP setting. In sum, the modified gRNAs designed based on nuclease structure (e.g., mod2, mod4, and mod6, specifically mod4) showed improved gene editing efficiency. EXAMPLE 3: CRISPR Nuclease-Mediated Editing of PCSK9 Target Sequences in Primary Human Hepatocyte (PHH) Donor Cells Using Various RNA Guide Spacer Nucleotide Lengths This Example describes genomic editing of the PCSK9 gene using a variant CRISPR nuclease introduced into cells by mRNA with synthetic RNA guides of various lengths in primary human hepatocytes (PHH) donors. The synthetic RNA guides used with the CRISPR nuclease contained spacer lengths ranging from 16 nucleotides (nt) to 20 nt. On-target indels and/or off-target indels were determined and compared between cells transfected with RNA guides of different spacer lengths. Structural information of these RNA guides can be found in Table 7 below. RNA guides were designed and ordered from Integrated DNA Technologies (IDT). These guide RNAs have the mod4 modification pattern – contained one phosphorothioate (PS) bond at the 5’ end and three PS bonds + three 2’-O-methylated bases at the 3’ end of the guide. Spacer lengths were either 16 nt, 18 nt, or 20 nt, with truncations occurring at the 3’ end. The DR sequence (SEQ ID NO: 7) for these gRNAs is provided in Table 2 above. Table 7. Target and RNA Guide Sequences for PCSK9 Guide PAM Target NHP Spacer Sequence RNA Guide Sequence S H l U CG G U CG m
Attorney Docket No.: 063586-537001WO Guide PAM Target NHP Spacer Sequence RNA Guide Sequence Sequence Homology PCSK9- GCGCAGCGG Yes GCGCAGCGGUG C*UUGUUGUAUAUGUCCUU CG *m U CA G* U CA m U CA *m
mRNA electroporation in primary human hepatocyte (PHH) donor cells PHH cells from human donors (from ThermoFisher or Lonza) were thawed from liquid nitrogen quickly in a 37oC water bath. The cells were added to pre-warmed hepatocyte recovery media (ThermoFisher, CM7000) and centrifuged for 10 minutes. The cell pellet was resuspended in appropriate volume of hepatocyte plating Medium (Williams’ Medium E, ThermoFisher A1217601 supplemented with Hepatocyte Plating Supplement Pack (serum- containing), ThermoFisher CM3000). The cells were subjected to a trypan blue viability count using the Vi-CELL BLU cell counter. The desired number of viable cells were then washed in PBS and resuspended in P3 buffer + supplement (Lonza, V4SP-3096) and transfection enhancer oligo (final concentration of 4 µM). Resuspended cells were dispensed at 125,000 cells/reaction into Lonza 96-well electroporation plates. mRNA effector (1 mg/mL in water) encoding the CRISPR nuclease polypeptide of SEQ ID NO: 6 was mixed with synthetic RNA guides (1 mM in water) at a 1:1 volume ratio. mRNA/guide RNA mixtures were added to each reaction at a final mRNA concentration of 25 nM, 6.25 nM, 1.56 nM, 0.78 nM, 0.39 nM, 0.19 nM, 0.09 nM, or 0.05 nM. The plates were electroporated using an electroporation device (program DS-150, Lonza 4D-nucleofector).5 minutes following electroporation, pre-warmed Hepatocyte plating medium was added to each well and mixed very gently by pipetting. For each technical replicate plate, 125,000 cells of diluted nucleofected cells were plated into a pre- warmed collagen-coated 96-well plate (ThermoFisher) with wells containing Hepatocyte plating medium. The cells were then incubated in a 37oC incubator. The media was changed
Attorney Docket No.: 063586-537001WO to hepatocyte maintenance media (Williams’ Medium E, ThermoFisher A1217601 supplemented with William’s E medium Cell Maintenance Cocktail, ThermoFisher CM 4000) after the cells attached after 4 hours. After 3 days post electroporation, cells in the wells were harvested using Accutase and transferred to 96-well twin.tec® PCR plates (Eppendorf) and centrifuged. Media was flicked off and cells were resuspended in DNA extraction buffer (QuickExtractTM). Samples were then cycled in a PCR machine at 65°C for 15 min, 68°C for 15 min, 98°C for 10 min. Samples were then frozen at -20°C, and subsequently analyzed for PCSK9 indels and indels of in silico predicted off-target sites using Next Generation Sequencing (NGS) as described in Example 1. FIG.3A shows PCSK9 indels in a PHH donor following electroporation of the variant CRISPR nuclease and the selected modified RNA guides. The 18 nt spacer length guides showed significantly higher on-target activity at PCSK9, while 16 nt and 20 nt spacer length guides showed equivalent editing. FIG.3B shows indels at in silico predicted off- target sites for PCSK9 targets. The 18 nt spacer length guides showed significantly higher off-target activity at sites tested.20 nt spacer length guides showed significantly lower, but detectable levels of editing at off-targets, while 16 nt spacer length guides showed no off- target activity at sites tested. Guides with 18 nt spacers showed higher on-target and higher off-target activity for the PCSK9-targeting guides tested. Guides with 16 nt spacers showed equivalent on-target activity compared to 20 nt spacers, but significantly lower off-target activity across several predicted off-target sites. Off-target analysis of PCSK9-E1T3 guides with various spacer lengths in PHH To assess off-target activity at a larger scale, rhAmpSeqTM (Integrated DNA Technologies) was performed in PHH cells, targeting in silico predicted off-targets for a 20 nt spacer PCSK9-E1T3 guide. Briefly, gDNA was extracted from primary human hepatocytes using the Zymo Quick-DNA Miniprep Plus kit, quantified by high sensitivity DNA Qubit, and was then normalized to 8ng/uL with IDTE pH 8.0. rhAmp primer pools for the first round of PCR (PCR1) were resuspended in IDTE pH7.5 to variable 10X concentrations based on the number of primers in each pool. If the panel contained ≥ 500 primers, the 10X rhAmp primer pool was 50uM total. If the panel contained between 100 and 500 primers, the 10X rhAmp Primer Pool was 100nM of each primer. If the panel contained ≤ 100 primers, the 10X
Attorney Docket No.: 063586-537001WO rhAmp Primer Pool was 250nM of each primer. rhAmp index primers for the second round of PCR (PCR2) were resuspended in IDTE pH8.0 to a working concentration of 5uM. Next Generation Sequencing (NGS) samples were prepared by two rounds of PCR. Two technical replicates were analyzed per sample or negative. PCR1 was used to amplify genomic regions. Reactions then underwent a 1.5X solid-phase reversible immobilization (SPRI) to purify the product, removing unused primers and primer dimer. PCR2 was performed to add Illumina adapters and indices. Reactions then underwent a 1X SPRI to purify the product. Diluted product was then run on a 2% agarose gel for QC, checking the size of the product against the design provided by IDT. Reactions were then pooled by scaling the volume pooled to the number of amplicons in each product so that each amplicon was equally represented in the library pool. Library pools were then run on a high sensitivity D1000 Tapestation kit to obtain an average fragment length, quantified by high sensitivity DNA Qubit, and normalized to 0.8nM. The number of required reads was determined by multiplying the total number of amplicons in a library pool, the estimated cell equivalents of the input gDNA (given an estimation of 6pg gDNA per cell), and a coverage multiplier of ten. Libraries were then sequenced on a 500 Cycle NovaSeq 6000 SP Reagent Kit v1.5. For NGS analysis, the indel mapping function used a sample’s fastq file, the amplicon reference sequence, and the forward primer sequence. For each read, a kmer-scanning algorithm was used to calculate the edit operations (match, mismatch, insertion, deletion) between the read and the reference sequence. In order to remove small amounts of primer dimer present in some samples, the first 30 nt of each read was required to match the reference and reads where over half of the mapping nucleotides are mismatches were filtered out as well. All reads passing those filters were used for analysis, and reads were counted as an indel read if they contained an insertion or deletion. The QC standard for the minimum number of reads passing filters was 1,000. For each amplicon, indel ratios, referring to the percentage of NGS reads comprising indels, were calculated for each sample and its cognate non-targeting control (negative). Amplicons with potential off-target activity were flagged by comparing the sample to its negative and observing a difference in one or more of the following representations of the NGS data: 1. Indel ratio 2. Heatmap of the fraction of reads with a given indel length 3. Figure with the indel ratio per position on the y-axis and amplicon position on the x-axis
Attorney Docket No.: 063586-537001WO Additionally, a background-corrected indel ratio was calculated by removing all reads from the sample which contained indels that also appeared in its negative and by removing all reads from the sample which contained indels that did not overlap with a window 20bp upstream and 20bp downstream from the putative cut site. Amplicons with background- corrected indel ratios >0.05% were flagged. For each flagged amplicon, the list of the unique reads (hashmap) from a sample was compared to its negative to determine if there was true off-target activity or it the flag originated from sequencing noise, amplification errors, primer dimerization, or mispriming. Similar to what was observed using NGS at select targeted sites (FIGS.4A-4E), rhAmpSeqTM in PHH demonstrated higher off-target activity in samples treated with 18 nt spacer length guides compared to 16 nt and 20 nt lengths (Table 8). The gRNA having the 16-nt spacer showed higher on-target editing efficacy as compared with the gRNAs having the 18-nt or 20-nt spacer. A further reduction in off-target activity was observed at the 16 nt length compared to 20 nt (Table 8). Table 8. Off-target Indel Activity Measured by rhAmpSeq for PCSK9-E1T3 Target ID 16 nt spacer 18 nt spacer 20 nt spacer On-target 73.63% 69.88% 53.68%
EXAMPLE 4: Method for Determining Efficacy of a PCSK9 Gene Editing System with LNP-Based Delivery Approach on Humanized PCSK Transgenic Mice This example investigates the efficacy of a gene editing system comprising an mRNA encoding the CRISPR nuclease polypeptide of SEQ ID NO: 6 and a PCSK9 guide RNA, with varying spacer lengths (16-nt or 20-nt) with mod4 modifications. The mRNAs expressing the CRISPR nuclease polypeptide and the gRNA were loaded into lipid nanoparticles and administered to mice expressing human PCSK9 and lacking expression of mouse PCSK9 (Essalmani et al, Biol. Chem, 399(12):1363-1374, 2018). The lipid nanoparticles contain 46.3% cationic lipid 6-((2-hexyldecanoyl)oxy)-N-(6- ((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium, 9.4% phospholipid 1,2- Distearoyl-sn-glycerol-3-phosphocholine (DSPC), 42.7% cholesterol, and 1.6% PEG lipid 2-
Attorney Docket No.: 063586-537001WO [(polyethylene glycol)-2000]-N,N ditetradecylacetamide, and was formulated with a Molar N/P ratio of ~6. The LNPs was prepared according to the general procedures described in (Schoenmaker, IJPharm, 601:120586, 2021), the relevant disclosures of which are incorporated by reference herein for the subject matter and purpose referenced herein. The nitrogen:phosphate (N/P) ratio was calculated from the results of HPLC (for determination of aminolipid mol) and ribogreen (for determination of mol of total RNAs), as indicated by the formula below: ^^^ ^^^^^^^^^^ (^^^^) ^^^ ^^^^^ ^^^ (^^^^^^^^^) Mice of two treatment groups harboring the human PCSK9 transgene were dosed at the two dose levels with interim- and long-term endpoints as described in Table 9. The impact of guide RNA spacer length was examined in vivo. Table 9. Mice Treatment Groups Group mRNA Guide Delivery Anima Dose Admin Duration # RNA l # (mg/kg) Route Endpoint ar ar ar ar
The guide RNA sequences and modifications are provided in Example 2 above. Interim readouts for PCSK9 levels were done biweekly for the first month and then monthly thereafter (FIG.5). The interim results for this experiment demonstrate that modification 4 (mod4) is able to achieve PCSK9 lowering in vivo when used with guides of a
Attorney Docket No.: 063586-537001WO 20mer and a 16mer spacer in a dose-dependent manner. At study endpoint, terminal blood will be collected for endpoint assessment of PCSK9, LDL-C, and triglycerides. Liver tissue will be collected at necropsy for the assessment of editing by NGS. Results from these studies enable the assessment and optimization of a PCSK9 gene editing system as provided herein, including the assessment of dose dependency on editing levels, guide RNA modification approach, spacer length, and the physiological benefit of editing as it relates the therapeutically relevant biomarkers, and the long term durability. EXAMPLE 5: Method for Determining Efficacy of Guide Modifications with LNP- Based Delivery Approach on Wildtype C57BL/6 Mice This example investigates the efficacy of a gene editing system comprising an mRNA encoding the CRISPR nuclease polypeptide of SEQ ID NO: 6 and two guides that both target the murine Hao1 (mHao1) gene with varying end modifications (mod 2, 4, and 6). The mRNAs expressing the CRISPR nuclease and the gRNA targeting mHao1 are loaded into lipid nanoparticles (see descriptions in Example 4 above) and administered to wildtype mice. The guide sequences are provided in Table 10 below. Table 10. Target and RNA Guide Sequences for HAO1 Guides Mod RNA Guide Sequence CG C A CC C A
Spacer sequences of 20-nt is used as an example. Can be shortened to 16-18-nt. The mice treatment groups are provided in Table 11 below.
Attorney Docket No.: 063586-537001WO Table 11. Mice Treatment Groups Group mRNA Guide Delivery Animal Dose Admin Duration # RNA # (mg/kg) Route Endpoint
At the 1-week endpoint, liver editing and Hao1 levels were measured via NGS and capillary western blot (JESS Simple Western System). All of the modified gRNAs resulted in some levels of HAO1 gene editing in liver cells measured by NGS with mod4 gRNA showed the highest editing efficiency. FIG.6A. Westen blot analysis showed that the protein levels of HAO1 in liver cells were reduced and mod4 gRNA led to a significant reduction of HAO1 protein. FIG.6B. See also FIG.6C. Overall, the data show that varying the end modification of the guides drives differing levels of liver editing and concomitant protein knockdown in vivo. Results from these studies enable the assessment of guide modifications using the gene editing system as provided herein, including the assessment of in vivo editing efficiency and protein knockdown.
Attorney Docket No.: 063586-537001WO OTHER EMBODIMENTS All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. EQUIVALENTS While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
Attorney Docket No.: 063586-537001WO All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or
Attorney Docket No.: 063586-537001WO unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.