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WO2025250443A1 - Compositions d'arn guide divisé et procédés d'activation d'une protéine effectrice crispr-cas avec une séquence nucléotidique courte - Google Patents

Compositions d'arn guide divisé et procédés d'activation d'une protéine effectrice crispr-cas avec une séquence nucléotidique courte

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Publication number
WO2025250443A1
WO2025250443A1 PCT/US2025/030627 US2025030627W WO2025250443A1 WO 2025250443 A1 WO2025250443 A1 WO 2025250443A1 US 2025030627 W US2025030627 W US 2025030627W WO 2025250443 A1 WO2025250443 A1 WO 2025250443A1
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actnucleicacid
rna
crispr
caprna
virus
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Daniel A. Fletcher
Amy LYDEN
Amanda MERIWETHER
Sungmin Son
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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Publication of WO2025250443A1 publication Critical patent/WO2025250443A1/fr
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Definitions

  • a Sequence Listing is provided herewith as a Sequence Listing XML, “BERK- 527PRV_SEQLIST.xml” created on May 24, 2024 and having a size of 478,931 bytes.
  • the contents of the Sequence Listing XML are incorporated by reference herein in their entirety.
  • RNA targets shorter than 21 nt has been a challenge.
  • this lower-length limit of detection excludes many miRNA species.
  • An estimated 15.5% (411 of 2656) of documented human miRNAs are less than 21 nt long, but it is likely that there are more miRNAs in this range that have not yet been discovered by current technologies.
  • miRNAs continue to be identified as important biomarkers for disease diagnosis and monitoring, it becomes more important to be able to detect the full range of miRNAs and discover new miRNAs.
  • Cas13a is a CRISPR-Cas protein that has found multiple applications in molecular diagnostics and therapeutics.
  • Cas13a a class 2, type VI CRISPR-Cas system, complexes with a guide RNA (gRNA) to form a Cas13a ribonucleoprotein (RNP).
  • the gRNA contains a stem-loop region that mediates the interaction between the protein and gRNA, which includes a programmable 20-28nt seed region.
  • the Cas13a RNP binds to a target RNA that is complementary to the seed region, activating the RNP and triggering non-specific cleavage of single-stranded RNA (ssRNA).
  • ssRNA single-stranded RNA
  • assays use a quenched fluorescent reporter composed of a fluorophore linked to a quencher by a short ssRNA segment.
  • the Cas13a RNP When the Cas13a RNP is activated by target RNA, the RNP cleaves the ssRNAs of the reporters, unquenching the fluorophores and generating a fluorescent signal that increases over time.
  • the rate at which the signal increases corresponds to the number of active Cas13a RNPs and enables quantification of the target RNA's abundance in a sample.
  • Cas13a One useful feature of Cas13a is that it can be used without the need for RNA extraction and purification, reverse transcription, and amplification steps that are usually required in PCR-based diagnostics. This enables direct detection of many RNA targets, including viral genomic RNA and host mRNA at levels that are not influenced by losses and variability of RNA extraction and reverse transcription. However, detection of short RNAs ( ⁇ 21 nt) like miRNAs, has been a challenge.
  • Circulating miRNAs are important molecular diagnostic targets that can serve as biomarkers for disease diagnosis and progression.
  • Current methods for miRNA detection are error-prone.
  • PCR-based methods including RT-qPCR and digital droplet PCR (ddPCR) require careful design of primers and probes that accommodate both the small size of miRNA (17-26nt) and the sequence similarity within miRNA families.
  • ddPCR digital droplet PCR
  • the modifications involved in the reverse transcription to cDNA often introduce error and reduce the reliability and repeatability of the assay. Furthermore, these methods are restricted to known miRNAs.
  • RT-qPCR and ddPCR methods have a similar sensitivity, with a limit of detection ranging from 0.25 to 8 cDNA copies/uL for RT-qPCR and 1 .25 to 2 cDNA copies/uL for ddPCR.
  • the true limit of detection of miRNA copies depends on the efficiency of the reverse transcription step.
  • PCR-based strategies are limited by the efficiency and specificity of the RNA purification and reverse transcription steps.
  • Next-generation sequencing is an alternate strategy for identifying and quantifying miRNAs, with the added potential for miRNA discovery.
  • it is similarly flummoxed by the need to extract, purify, and reverse transcribe miRNAs.
  • Substantial error and bias can be introduced during library preparation when RNA is reverse transcribed to cDNA and amplified.
  • split guide RNA methods and compositions for activating a CRISPR-Cas effector protein with a nucleic acid of interest. This includes contacting a capture nucleic acid (capNucleicAcid) with a CRISPR-Cas effector protein and a CRISPR-Cas guide RNA in the presence of a nucleic acid of interest (referred to as an activating nucleic acid (actNucleicAcid)) (e.g., a DNA of interest/activating DNA (actDNA) or an RNA of interest/activating RNA (actRNA)).
  • an activating nucleic acid e.g., a DNA of interest/activating DNA (actDNA) or an RNA of interest/activating RNA (actRNA)
  • compositions and methods can provide an alternate strategy to detect short nucleic acid sequences, e.g., RNAs, e.g., ⁇ 21 nucleotides (nt) (e.g., miRNAs) that does not use the traditional paradigm of CRISPR-Cas-based nucleic acid detection. Instead, the guide RNA (gRNA) is split (see, e.g., FIG.
  • an “activating nucleic acid” or “actNucleicAcid” acts as if it were the 3’ end of a traditional guide RNA.
  • the sequence of the actNucleicAcid being detected is small (e.g., can detect short RNAs such as miRNAs) with lengths as small as 8 nt.
  • the guide RNA has a shorter targeting sequence (guide sequence - also referred to herein as a seed sequence) than what is normally used with CRISPR-Cas systems, and the actNucleicAcid (e.g., ‘activating RNA’) acts as the remainder of the guide sequence - thus, the guide RNA has been split into two parts: a guide RNA with a truncated guide sequence, and an actNucleicAcid.
  • the capNucleicAcid e.g., a capture RNA (capRNA) or capture DNA (capDNA)
  • the capNucleicAcid includes two regions: an anchor region that hybridizes to the guide RNA, and a capture region, which hybridizes to the actNucleicAcid.
  • the capNucleicAcid provides a landing pad - a sequence that can be targeted by traditional CRISPR-Cas systems - however, the guide sequence of the guide RNA (the split guide RNA) only hybridizes to part of that targeted sequence - the actNucleicAcid hybridizes to the remainder.
  • this strategy uses a CRISPR-Cas effector protein (e.g., a Cas13 protein, a Cas12 protein, and the like, e.g., a Cas13a protein), a split gRNA, and a sequencespecific capture nucleic acid (capNucleicAcid) (e.g., a capture RNA (capRNA) or capture DNA (capDNA)) to detect the actNucleicAcid.
  • a CRISPR-Cas effector protein e.g., a Cas13 protein, a Cas12 protein, and the like, e.g., a Cas13a protein
  • a split gRNA e.g., a split gRNA
  • a sequencespecific capture nucleic acid (capNucleicAcid) e.g., a capture RNA (capRNA) or capture DNA (capDNA)
  • the split gRNA and actNucleicAcid hybridize to (e.g., are fully complemented by) the capNucleicAcid (e.g., capRNA), which plays the role of a targeted nucleic acid (e.g., RNA or DNA) in a traditional CRISPR-Cas system.
  • the capNucleicAcid e.g., capRNA
  • the portion of the capNucleicAcid that hybridizes to the split gRNA is referred to as the “anchor region”, and the portion of the capNucleicAcid that hybridizes to the actNucleicAcid is referred to as the “capture region” (see FIG. 2A).
  • actNucleicAcid e.g., actRNA or actDNA
  • the CRISPR-Cas effector protein is not activated.
  • the CRISPR-Cas effector protein is activated.
  • a subject CRISPR-Cas effector protein has transcleavage activity (also referred to in the art as collateral cleavage activity).
  • the trans-cleavage activity of the CRISPR-Cas effector protein is activated.
  • the present disclosure provides a method of detecting a nucleic acid (e.g., RNA or DNA) in a sample.
  • a nucleic acid e.g., RNA or DNA
  • the sample is a cell-free sample.
  • the sample comprises cells.
  • the sample comprises a cell lysate.
  • the sequences of the split gRNA and the anchor region of the capNucleicAcid e.g., capRNA, capDNA
  • the capture region of the capNucleicAcid that can be changed such that it includes a sequence that is perfectly complementary to the desired actNucleicAcid.
  • a given sample includes the desired actNucleicAcid (e.g., a particular miRNA of interest)
  • the split gRNA, the capNucleicAcid, the CRISPR-Cas effector protein, e.g., Cas13a protein will lead to activation of the trans cleavage activity of the CRISPR-Cas effector protein.
  • the CRISPR-Cas effector protein can then cleave the detector nucleic acid to generate a detectable signal.
  • the split guide system extends the lower-length range of detectable sequences to 8nt, while maintaining the sensitivity and specificity of a full-length guide system.
  • FIG. 1A-1D demonstrate Cas13a is activated by ssRNA ⁇ 20 nucleotides in length with a split gRNA.
  • FIG. 2A-2E demonstrate Cas13a detection of 10 nucleotide ssRNA with a split guide system.
  • FIG. 3A-3C demonstrate activity of a split guide system across a range of RNA lengths and sequences.
  • FIG. 4A-4C demonstrate that activity of a split guide system is specific against RNA mismatches and misalignments.
  • FIG. 5A-5F demonstrate detection of endogenous cellular miRNA with a split guide system.
  • FIG. 6A-6B demonstrate detection of target RNAs of varying lengths and at varying concentrations with a split guide system.
  • FIG. 7A-7D depict activity of a split guide system with varying concentrations, species, and lengths of capRNAs.
  • FIG. 8 depicts predicted structures for a split gRNA-capRNA complex with varying capRNAs.
  • FIG. 9A-9D depict activity of a split guide system with various combinations of anchor lengths, actRNA lengths, and capRNA lengths.
  • FIG. 10A-10E depict a comparison of two sequence-unique split guide systems.
  • FIG. 11 A-11 B demonstrate mismatch sensitivity of a split guide system and detection of target actRNA in a total cell RNA background, respectively.
  • FIG. 12 depicts a histogram of showing the length distribution of validated human miRNAs.
  • FIG. 13 depicts predicted structures for a split gRNA-capRNA complex with varying capRNAs targeting different cellular miRNAs.
  • FIG. 14A-14C demonstrate detection of Influenza A viral RNA with a split guide system.
  • polynucleotide and nucleic acid refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • terms “polynucleotide” and “nucleic acid” encompass single-stranded DNA; double-stranded DNA; multi-stranded DNA; single-stranded RNA; double-stranded RNA; multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • hybridizable or “complementary” or “substantially complementary” it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that allows it to non-covalently bind, i.e. form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength.
  • a nucleic acid e.g. RNA, DNA
  • anneal i.e. form Watson-Crick base pairs and/or G/U base pairs
  • Standard Watson-Crick base-pairing includes: adenine/adenosine) (A) pairing with thymidine/thymidine (T), A pairing with uracil/ uridine (U), and guanine/guanosine) (G) pairing with cytosine/cytidine (C).
  • A adenine/adenosine
  • T thymidine/thymidine
  • U uracil/ uridine
  • G guanine/guanosine
  • C cytosine/cytidine
  • G cytosine/cytidine
  • G can also base pair with U.
  • G/U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA.
  • a G e.g., of a proteinbinding segment (e.g., dsRNA duplex) of a guide RNA molecule; of a target nucleic acid (e.g., target DNA) base pairing with a guide RNA
  • a G e.g., of a proteinbinding segment (e.g., dsRNA duplex) of a guide RNA molecule; of a target nucleic acid (e.g., target DNA) base pairing with a guide RNA
  • a target nucleic acid e.g., target DNA
  • a G/U base-pair can be made at a given nucleotide position of a protein-binding segment (e.g., dsRNA duplex) of a guide RNA molecule, the position is not considered to be non- complementary, but is instead considered to be complementary.
  • a protein-binding segment e.g., dsRNA duplex
  • Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible.
  • the conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences.
  • the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).
  • sequence of a polynucleotide need not be 100% complementary (i.e., perfect complementarity) to that of its target nucleic acid to be specifically hybridizable - although in some embodiments nucleic acids are 100% complementary to one another.
  • a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a ‘bulge’, and the like).
  • a polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize.
  • an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity.
  • the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides.
  • Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method.
  • Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981 , 2, 482-489).
  • peptide refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • Binding refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid; between a guide RNA and a target nucleic acid; and the like). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner).
  • Binding interactions are generally characterized by a dissociation constant (Kd) of less than 10 6 M, less than 10 7 M, less than 10 8 M, less than 10 9 M, less than 10 10 M, less than 10 11 M, less than 10 12 M, less than 10 13 M, less than 10 14 M, or less than 10 15 M.
  • Kd dissociation constant
  • Affinity refers to the strength of binding, increased binding affinity being correlated with a lower K d .
  • binding domain it is meant a protein domain that is able to bind non-covalently to another molecule.
  • a binding domain can bind to, for example, an RNA molecule (an RNA-binding domain) and/or a protein molecule (a protein-binding domain).
  • RNA-binding domain an RNA-binding domain
  • protein-binding domain a protein molecule
  • it can in some cases bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more regions of a different protein or proteins.
  • a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide containing side chains consisting of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; a group of amino acids having acidic side chains consists of glutamate and aspartate; and a group of amino acids having sulfur containing side chains consists of cysteine and methionine.
  • Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysinearginine, alanine-valine-glycine, and asparagine-glutamine.
  • Coded amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F); proline (Pro; P), serine (Ser; S), threonine (Thr; T), try
  • a polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different ways.
  • sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, Phyre2, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/, http://www.sbg.bio.ic.ac.uk/ ⁇ phyre2/. See, e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.
  • DNA regulatory sequences refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence (e.g., protein coding) and/or regulate translation of an encoded polypeptide.
  • a non-coding sequence e.g., guide RNA
  • a coding sequence e.g., protein coding
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence.
  • Eukaryotic promoters will often, but not always, contain "TATA” boxes and “CAT” boxes.
  • Various promoters, including inducible promoters, may be used to drive the various nucleic acids (e.g., vectors) of the present disclosure.
  • nucleic acid refers to a nucleic acid, polypeptide, cell, or organism that is found in nature.
  • Recombinant means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems.
  • DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system.
  • Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see “DNA regulatory sequences", above). Alternatively, DNA sequences encoding RNA (e.g., guide RNA) that is not translated may also be considered recombinant. Thus, e.g., the term "recombinant" nucleic acid refers to one which is not naturally occurring, e.g, is made by the artificial combination of two otherwise separated segments of sequence through human intervention.
  • This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a codon encoding the same amino acid (e.g., codon optimization), a conservative amino acid, or a non-conservative amino acid. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • a recombinant polynucleotide encodes a polypeptide
  • the sequence of the encoded polypeptide can be naturally occurring (“wild type”) or can be a variant (e.g., a mutant) of the naturally occurring sequence.
  • the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose sequence does not naturally occur.
  • a “recombinant” polypeptide is encoded by a recombinant DNA sequence, but the sequence of the polypeptide can be naturally occurring (“wild type”) or non-naturally occurring (e.g., a variant, a mutant, etc.).
  • a "recombinant” polypeptide is the result of human intervention, but may have a naturally occurring amino acid sequence.
  • a "vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an “insert”, may be attached so as to bring about the replication of the attached segment in a cell.
  • An “expression cassette” comprises a DNA coding sequence operably linked to a promoter.
  • "Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression (the coding sequence can also be said to be operably linked to the promoter).
  • recombinant expression vector or “DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and one insert.
  • Recombinant expression vectors are usually generated for the purpose of expressing and/or propagating the insert(s), or for the construction of other recombinant nucleotide sequences.
  • the insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.
  • Heterologous refers to a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively.
  • a CRISPR-Cas effector protein can be fused to an active domain from a non- CRISPR-Cas effector protein (e.g., a histone deacetylase), and the sequence of the active domain could be considered a heterologous polypeptide (it is heterologous to the CRISPR-Cas effector protein).
  • a guide sequence of a guide RNA can be heterologous to the protein-binding sequence (a scaffold) of the guide RNA - as such, the guide sequence is not found in nature together with the protein-binding sequence (the scaffold).
  • split guide RNA methods and compositions for activating a CRISPR-Cas effector protein with a nucleic acid of interest. This includes contacting a capture nucleic acid (capNucleicAcid) with a CRISPR-Cas effector protein and a CRISPR-Cas guide RNA in the presence of a nucleic acid of interest (referred to as an activating nucleic acid (actNucleicAcid)) (e.g., a DNA of interest/activating DNA (actDNA) or an RNA of interest/activating RNA (actRNA)).
  • an activating nucleic acid e.g., a DNA of interest/activating DNA (actDNA) or an RNA of interest/activating RNA (actRNA)
  • compositions and methods can provide an alternate strategy to detect short nucleic acid sequences, e.g., RNAs, e.g., ⁇ 21 nucleotides (nt) (e.g., miRNAs) that does not use the traditional paradigm of CRISPR-Cas-based nucleic acid detection. Instead, the guide RNA (gRNA) is split (see, e.g., FIG.
  • RNAs e.g., ⁇ 21 nucleotides (nt) (e.g., miRNAs)
  • gRNA guide RNA
  • an “activating nucleic acid” or “actNucleicAcid” acts as if it were the 3’ end of a traditional guide RNA.
  • the sequence of the actNucleicAcid being detected is small (e.g., can detect short RNAs such as miRNAs) with lengths as small as 8 nt.
  • the guide RNA has a shorter targeting sequence (guide sequence - also referred to herein as a seed sequence) than what is normally used with CRISPR-Cas systems, and the actNucleicAcid (e.g., ‘activating RNA’) acts as the remainder of the guide sequence - thus, the guide RNA has been split into two parts: a guide RNA with a truncated guide sequence, and an actNucleicAcid.
  • the capNucleicAcid e.g., a capture RNA (capRNA) or capture DNA (capDNA)
  • the capNucleicAcid includes two regions: an anchor region that hybridizes to the guide RNA, and a capture region, which hybridizes to the actNucleicAcid.
  • the capNucleicAcid provides a landing pad - a sequence that can be targeted by traditional CRISPR-Cas systems - however, the guide sequence of the guide RNA (the split guide RNA) only hybridizes to part of that targeted sequence - the actNucleicAcid hybridizes to the remainder.
  • this strategy uses a CRISPR-Cas effector protein (e.g., a Cas13 protein, a Cas12 protein, and the like, e.g., a Cas13a protein), a split gRNA, and a sequencespecific capture nucleic acid (capNucleicAcid) (e.g., a capture RNA (capRNA) or capture DNA (capDNA)) to detect the actNucleicAcid.
  • a CRISPR-Cas effector protein e.g., a Cas13 protein, a Cas12 protein, and the like, e.g., a Cas13a protein
  • a split gRNA e.g., a split gRNA
  • a sequencespecific capture nucleic acid (capNucleicAcid) e.g., a capture RNA (capRNA) or capture DNA (capDNA)
  • the split gRNA and actNucleicAcid hybridize to (e.g., are fully complemented by) the capNucleicAcid (e.g., capRNA), which plays the role of a targeted nucleic acid (e.g., RNA or DNA) in a traditional CRISPR-Cas system.
  • the capNucleicAcid e.g., capRNA
  • the portion of the capNucleicAcid that hybridizes to the split gRNA is referred to as the “anchor region”, and the portion of the capNucleicAcid that hybridizes to the actNucleicAcid is referred to as the “capture region” (see FIG. 2A).
  • the actNucleicAcid e.g., actRNA or actDNA
  • the CRISPR-Cas effector protein is not activated.
  • the CRISPR-Cas effector protein is activated.
  • a subject CRISPR-Cas effector protein has transcleavage activity (also referred to in the art as collateral cleavage activity).
  • the trans-cleavage activity of the CRISPR-Cas effector protein is activated.
  • the present disclosure provides a method of detecting a nucleic acid (e.g., RNA or DNA) in a sample.
  • a nucleic acid e.g., RNA or DNA
  • the sample is a cell-free sample.
  • the sample comprises cells.
  • the sample comprises a cell lysate.
  • the capture region of the capNucleicAcid that can be changed such that it includes a sequence that is perfectly complementary to the desired actNucleicAcid. If a given sample includes the desired actNucleicAcid (e.g., a particular miRNA of interest), contacting the sample with the system (the split gRNA, the capNucleicAcid, the CRISPR-Cas effector protein, e.g., Cas13a protein) will lead to activation of the trans cleavage activity of the CRISPR-Cas effector protein. When a detector nucleic acid is also included, the CRISPR-Cas effector protein can then cleave the detector nucleic acid to generate a detectable signal.
  • the split guide system extends the lower-length range of detectable sequences to 8nt, while maintaining the sensitivity and specificity of a full-length guide system.
  • a capture Nucleic Acid includes an anchor region and a capture region.
  • the anchor region hybridizes to the guide RNA (the guide sequence of the guide RNA), while the capture region hybridizes to the nucleic acid of interest (actNucleicAcid).
  • actNucleicAcid the nucleic acid of interest
  • the anchor region can remain constant, as can the guide sequence of the guide RNA - it is the capture region that is changed in order to hybridize to (or ‘capture’) to different nucleic acid targets of interest (actNucleicAcids).
  • the capture region is positioned 5’ of the anchor region (see, e.g., FIG. 2A). In some cases, the capture region is positioned 3’ of the anchor region.
  • the capNucleicAcid is an RNA (capRNA), e.g., a single stranded RNA (ssRNA).
  • the capNucleicAcid is a DNA (capDNA), e.g., a single stranded DNA (ssDNA).
  • the capNucleicAcid is a double stranded DNA (dsDNA) (e.g., a Cas12a protein can recognize dsDNA as a target). In such cases, a PAM sequence can be taken into account and included into the capDNA.
  • the anchor region of the capNucleicAcid is 9-12 nt long (e.g., 9-11 , 9-10, 10-12, 10-11 , 11-12 nt). In some cases, the anchor region of the capNucleicAcid (e.g., capRNA) is 10-11 nt long. In some cases, the anchor region of the capNucleicAcid (e.g., capRNA) is 9 nt long. In some cases, the anchor region of the capNucleicAcid (e.g., capRNA) is 10 nt long. In some cases, the anchor region of the capNucleicAcid (e.g., capRNA) is 11 nt long. In some cases, the anchor region of the capNucleicAcid (e.g., capRNA) is 12 nt long.
  • the capture region of the capNucleicAcid is 10-21 nt long (e.g., 10-20, 10-18, 10-16, 10-15, 10-14, 10-12, 12-21 , 12-20, 12-18, 12-16, 12-15, 12-14, 14-21 , 14-20, 14-18, 14-16, 14-15, 10-20, 10-18, 10-16, 10-15, IQ- 14, 10-12 nt).
  • the capture region of the capNucleicAcid e.g., capRNA
  • the capture region of the capNucleicAcid is about 10 nt long.
  • the capture region of the capNucleicAcid is about 15 nt long.
  • the capture region of the capNucleicAcid is about 20 nt long.
  • the capNucleicAcid can be any convenient length.
  • the capNucleicAcid e.g., capRNA
  • the capNucleicAcid is 20-45 nt long (e.g., 20-40, 20- 35, 20-30, 20-25, 25-45, 25-40, 25-35, 25-30, 30-45, 30-40, 30-35, 35-45, or 35-40 nt).
  • the capNucleicAcid e.g, capRNA
  • the capNucleicAcid is 30-40 nt long (e.g., 30-35 or 35-40 nt).
  • a subject capNucleicAcid can include a 5’ tail region (e.g., in some cases positioned 5’ of the capture region).
  • the 5’ tail region is 1-12 nt long (e.g., 1 -10, 1 -8, 1-6, 1 -5, 1-3, 2-12, 2-10, 2-8, 2-6, 4-12, 4-10, 4-8, 4-5, 5-12, 5-10, 5-8, 7-12, 7-10, 7-8, 8-12, 8-10, 10-12 nt).
  • the 5’ tail region is 1-12 nt long (e.g., 1 -10, 1 -8, 1-6, 1 -5, 1-3, 2-12, 2-10, 2-8, 2-6, 4-12, 4-10, 4-8, 4-5, 5-12, 5-10, 5-8, 7-12, 7-10, 7-8, 8-12, 8-10, 10-12 nt).
  • the 5’ tail region is 1-12 nt long (e.g., 1 -10, 1 -8, 1-6, 1 -5,
  • a subject capNucleicAcid (e.g., capRNA) can include a 3’ region (e.g., in some cases positioned 3’ of the anchor region).
  • the 3’ region is 1-10 nt long (e.g., 1 -8, 1 -6, 1-5, 1 -3, 2-10, 2-8, 2-6, 2-4, 3-10, 3-8, 3-6, 3-5, 4-10, 4-8, 4-6,
  • the 3’ region is 3-5 nt long. In some cases, the 3’ region is 3 nt long. In some cases, the 3’ region is 4 nt long. In some cases, the 3’ region is 5 nt long.
  • a nucleic acid that binds to and thereby forms a ribonucleoprotein (RNP) complex with a CRISPR-Cas effector protein and targets the complex to a specific location within a target nucleic acid is referred to herein as a “guide RNA” or “CRISPR-Cas guide nucleic acid” or “CRISPR-Cas guide RNA” or simply a “guide.”
  • guide RNA or “CRISPR-Cas guide nucleic acid” or “CRISPR-Cas guide RNA” or simply a “guide.”
  • a guide RNA can be referred to by the protein to which it corresponds.
  • the corresponding guide RNA can be referred to as a “Cas12 guide RNA.”
  • the corresponding guide RNA can be referred to as a “Cas13 guide RNA.”
  • a subject guide RNA e.g., a Cas13 guide RNA, a Cas12 guide RNA
  • a Cas13 guide RNA also in some cases referred to as a “crRNA”, e.g., Cas13 crRNA, Cas12 crRNA
  • includes a “guide sequence” also referred to as a spacer, or a targeting sequence
  • a “scaffold” also referred to as a direct repeat (DR), handle, protein-binding region, or constant region).
  • the scaffold is 5’ or 3’ of the guide sequence, depending
  • the guide sequence of has complementarity with (hybridizes to) a target sequence of a target nucleic acid.
  • the target sequence with which the guide sequence hybridizes is the anchor region of the capNucleicAcid (e.g., capRNA or capDNA).
  • the base of the target RNA that is immediately 3’ of the target sequence (protospacer) is not a G.
  • the three bases of the target nucleic acid that are immediately 3’ of the target sequence (protospacer) are NAN or NNA, where N is any nucleotide.
  • the base of the target sequence that is immediately 5’ of the target sequence (protospacer) is A, U, or G.
  • the guide sequence of a subject split guide RNA system is in general shorter than the guide sequence of a standard guide RNA (which is usually about 20 nt long).
  • the guide sequence is 8-15 nucleotides (nt) long (e.g., 8-14, 8- 13, 8-12, 8-11 , 8-10, 8-9, 9-15, 9-14, 9-13, 9-12, 9-11 , 9-10, 10-15, 10-14, 10-13, 10-12, 10-11 , 11-15, 11 -14, 11-13, 11 -12, 12-15, 12-14, 12-13, 13-15, 13-14, or 14- 15 nt).
  • the guide sequence is 9-11 nt long (e.g., 9-10, 10-11 nt).
  • the guide sequence is 9 nt long.
  • the guide sequence is 10 nt long.
  • the guide sequence is 11 nt long.
  • the guide sequence has 80% or more (e.g., 85% or more, 90% or more, 95% or more, or 100% complementarity) with the target sequence of the anchor region of the capNucleicAcid. In some cases, the guide sequence is 100% complementary to the anchor region of the capNucleicAcid.
  • crRNA repeat sequences also known as the scaffold
  • Cas12a proteins include:
  • AAUUUCUACUAUUGUAGAU 3’ (SEQ ID NO: 249) - [spacer] MbCasI 2a/Mb2Cas12a/Mb3Cas12a crRNA:
  • the scaffold of a guide RNA comprises a nucleotide sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%) sequence identity with the nucleotide sequence of SEQ ID NO: 246. In some cases, the scaffold of a guide RNA comprises a nucleotide sequence having 85% or more (e.g., 90% or more, 95% or more, or 100%) sequence identity with the nucleotide sequence of SEQ ID NO: 246.
  • the scaffold of a guide RNA comprises a nucleotide sequence having 95% or more (e.g., 100%) sequence identity with the nucleotide sequence of SEQ ID NO: 246. In some cases, the scaffold of a guide RNA comprises the nucleotide sequence of SEQ ID NO: 246.
  • the scaffold of a guide RNA comprises a nucleotide sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%) sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 246-252. In some cases, the scaffold of a guide RNA comprises a nucleotide sequence having 85% or more (e.g., 90% or more, 95% or more, or 100%) sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 246-252.
  • the scaffold of a guide RNA comprises a nucleotide sequence having 95% or more (e.g., 100%) sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 246-252. In some cases, the scaffold of a guide RNA comprises the nucleotide sequence of any one of SEQ ID NOs: 246- 252.
  • the scaffold of a guide RNA is 15 or more nucleotides (nt) in length (e.g., 18 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more nt, 32 or more, 33 or more, 34 or more, or 35 or more nt in length).
  • the scaffold of a guide RNA is 18 or more nt in length.
  • the scaffold of a guide RNA has a length in a range of from 12 to 100 nt (e.g., from 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40, 15 to 100, 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 20, 19 to 100, 19 to 90, 19 to 80, 19 to 70, 19 to 60, 19 to 50, 19 to 40, 19 to 30, 19 to 20,
  • the scaffold of a guide RNA has a length in a range of from 18-22 nt. In some cases, the scaffold of a guide RNA has a length in a range of from 19-20 nt.
  • the scaffold of a guide RNA is truncated relative to (shorter than) the corresponding region of a corresponding wild type guide RNA. In some cases, the scaffold of a guide RNA is extended relative to (longer than) the corresponding region of a corresponding wild type guide RNA.
  • a subject guide RNA is 30 or more nucleotides (nt) in length (e.g., 34 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, or 80 or more nt in length). In some cases, the guide RNA is 35 or more nt in length. In some cases, a subject guide RNA is 30-60 nt (e.g., 30-50, 30-45, 30-40, , 35-60, 35-50, 35-45, 35-40, 40-60, 40-50, or 40-45 nt) in length.
  • the following sequences are each an example of a scaffold of a naturally existing Cas13a guide RNA (e.g., a scaffold that is 5’ of the guide sequence) (See, e.g., Feng et al., Anal Chem. 2023 Jan 10;95(1 ) :206-217):
  • a subject Cas13 guide RNA includes a nucleotide sequence having 70% or more identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% identity) with the sequence set forth in any one of SEQ ID NOs:226-232.
  • a subject Cas13 guide RNA includes a nucleotide sequence having 90% or more identity (e.g., 95% or more, 98% or more, 99% or more, or 100% identity) with the sequence set forth in any one of SEQ ID NOs:245-248.
  • a subject Cas13 guide RNA includes the nucleotide sequence set forth in any one of SEQ ID NOs: 226- 232.
  • a subject Cas13 guide RNA includes a nucleotide sequence having 70% or more identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% identity) with the sequence set forth in SEQ ID NO:228.
  • a subject Cas13 guide RNA includes a nucleotide sequence having 90% or more identity (e.g., 95% or more, 98% or more, 99% or more, or 100% identity) with the sequence set forth in SEQ ID NO:228.
  • a subject Cas13 guide RNA includes the nucleotide sequence set forth in SEQ ID NO:228.
  • a subject Cas13 guide RNA includes a nucleotide sequence having 70% or more identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% identity) with the sequence set forth in SEQ ID NO:229.
  • a subject Cas13 guide RNA includes a nucleotide sequence having 90% or more identity (e.g., 95% or more, 98% or more, 99% or more, or 100% identity) with the sequence set forth in SEQ ID NO:229.
  • a subject Cas13 guide RNA includes the nucleotide sequence set forth in SEQ ID NO:229.
  • the constant region of a Cas13 guide RNA is 15 or more nucleotides (nt) in length (e.g., 18 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more nt, 32 or more, 33 or more, 34 or more, or 35 or more nt in length).
  • the constant region of a Cas13 guide RNA is 29 or more nt in length.
  • the constant region of a Cas13 guide RNA has a length in a range of from 12 to 100 nt (e.g., from 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40, 15 to 100, 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 25 to 100, 25 to 90, 25 to 80, 25 to 70, 25 to 60, 25 to 50, 25 to 40, 28 to 100, 28 to 90, 28 to 80, 28 to 70, 28 to 60, 28 to 50, 28 to 40, 29 to 100, 29 to 90, 29 to 80, 29 to 70, 29 to 60, 29 to 50, or 29 to 40 nt).
  • 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40 15 to 100, 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to
  • the constant region of a Cas13 guide RNA has a length in a range of from 28 to 100 nt. In some cases, the constant region of a Cas13 guide RNA has a length in a range of from 28 to 40 nt.
  • the constant region is truncated relative to (shorter than) the corresponding region of a corresponding wild type Cas13 guide RNA.
  • the mature LseCas13 guide RNA can include a constant region that is 30 nucleotides (nt) in length, and a subject truncated Cas13 guide RNA (relative to the Lse Cas13 guide RNA) can therefore have a constant region that is less than 30 nt in length (e.g., less than 29, 28, 27, 26, 25, 22, or 20 nt in length).
  • a truncated Cas13 guide RNA includes a constant region that has a length in a range of from 12 to 29 nt (e.g., from 12 to 28, 12 to 27, 12 to 26, 12 to 25, 12 to 22, 12 to 20, 12 to 18 nt, 14 to 29, 14 to 28, 14 to 27, 14 to 26, 14 to 25, 14 to 22, 14 to 20, 14 to 18 nt, 16 to 29, 16 to 28, 16 to 27, 16 to 26, 16 to 25, 16 to 22, 16 to 20, 16 to 18).
  • the truncated Cas13 guide RNA is truncated by one or more nt (e.g., 2 or more, 3 or more, 4 or more, 5 or more, or 10 or more nt), e.g., relative to a corresponding wild type Cas13 guide).
  • nt e.g., 2 or more, 3 or more, 4 or more, 5 or more, or 10 or more nt
  • the constant region of the Cas13 guide RNA is extended relative to (longer than) the corresponding region of a corresponding wild type Cas13 guide RNA.
  • a mature LseCas13 guide RNA can include a constant region that is 30 nucleotides (nt) in length, and an extended Cas13 guide RNA (relative to the LseCas13 guide RNA) can therefore have a constant region that is longer than 30 nt (e.g., longer than 31 , longer than 32, longer than 33, longer than 34, or longer than 35 nt).
  • an extended Cas13 guide RNA includes a constant region that has a length in a range of from 30 to 100 nt (e.g., from 30 to 90, 30 to 80, 30 to 70, 30 to 60, 30 to 50, or 30 to 40 nt).
  • the extended Cas13 guide RNA includes a constant that is extended (e.g., relative to the corresponding region of a corresponding wild type Cas13 guide RNA) by one or more nt (e.g., 2 or more, 3 or more, 4 or more, 5 or more, or 10 or more nt).
  • the constant region of a Cas13 guide RNA is 15 or more nucleotides (nt) in length (e.g., 18 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more nt, 32 or more, 33 or more, 34 or more, or 35 or more nt in length).
  • the constant region of a Cas13 guide RNA is 29 or more nt in length.
  • the constant region of a Cas13 guide RNA has a length in a range of from 12 to 100 nt (e.g., from 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40, 15 to 100, 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 25 to 100, 25 to 90, 25 to 80, 25 to 70, 25 to 60, 25 to 50, 25 to 40, 28 to 100, 28 to 90, 28 to 80, 28 to 70, 28 to 60, 28 to 50, 28 to 40, 29 to 100, 29 to 90, 29 to 80, 29 to 70, 29 to 60, 29 to 50, or 29 to 40 nt).
  • 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40 15 to 100, 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to
  • the constant region of a Cas13 guide RNA has a length in a range of from 28 to 100 nt. In some cases, the constant region of a Cas13 guide RNA has a length in a range of from 28 to 40 nt.
  • a subject Cas13 guide RNA is 30 or more nucleotides (nt) in length (e.g., 34 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, or 80 or more nt in length). In some cases, the Cas13 guide RNA is 35 or more nt in length.
  • a subject Cas13 guide RNA has a length in a range of from 30 to 120 nt (e.g., from 30 to 110, 30 to 100, 30 to 90, 30 to 80, 30 to 70, 30 to 60, 35 to 120, 35 to 110, 35 to 100, 35 to 90, 35 to 80, 35 to 70, 35 to 60, 40 to 120, 40 to 110, 40 to 100, 40 to 90, 40 to 80, 40 to 70, 40 to 60, 50 to 120, 50 to 110, 50 to 100, 50 to 90, 50 to 80, or 50 to 70 nt).
  • the Cas13 guide RNA has a length in a range of from 33 to 80 nt.
  • the Cas13 guide RNA has a length in a range of from 35 to 60 nt.
  • a subject Cas13 guide RNA is truncated relative to (shorter than) a corresponding wild type Cas13 guide RNA.
  • a mature Lse Cas13 guide RNA can be 50 nucleotides (nt) in length, and a truncated Cas13 guide RNA (relative to the Lse Cas13 guide RNA) can therefore in some cases be less than 50 nt in length (e.g., less than 49, 48, 47, 46, 45, 42, or 40 nt in length).
  • a truncated Cas13 guide RNA has a length in a range of from 30 to 49 nt (e.g., from 30 to 48, 30 to 47, 30 to 46, 30 to 45, 30 to 42, 30 to 40, 35 to 49, 35 to 48, 35 to 47, 35 to 46, 35 to 45, 35 to 42, or 35 to 40 nt).
  • the truncated Cas13 guide RNA is truncated by one or more nt (e.g., 2 or more, 3 or more, 4 or more, 5 or more, or 10 or more nt), e.g., relative to a corresponding wild type Cas13 guide).
  • a subject Cas13 guide RNA is extended relative to (longer than) a corresponding wild type Cas13 guide RNA.
  • a mature Lse Cas13 guide RNA can be 50 nucleotides (nt) in length, and an extended Cas13 guide RNA (relative to the Lse Cas13 guide RNA) can therefore in some cases be longer than 50 nt (e.g., longer than 51 , longer than 52, longer than 53, longer than 54, or longer than 55 nt).
  • an extended Cas13 guide RNA has a length in a range of from 51 to 100 nt (e.g., from 51 to 90, 51 to 80, 51 to 70, 51 to 60, 53 to 100, 53 to 90, 53 to 80, 53 to 70, 53 to 60, 55 to 100, 55 to 90, 55 to 80, 55 to 70, or 55 to 60 nt).
  • the extended Cas13 guide RNA is extended (e.g., relative to a corresponding wild type Cas13 guide RNA) by one or more nt (e.g., 2 or more, 3 or more, 4 or more, 5 or more, or 10 or more nt).
  • the guide RNA can be introduced into a cell as an RNA (or as a DNA/RNA hybrid) or can be introduced as a nucleic acid encoding the RNA (e.g., a DNA such as an expression vector such as a viral or plasmid DNA), in which case the cell transcribes the RNA from the introduced DNA.
  • the nucleotide sequence encoding the guide RNA is operably linked to a promoter (e.g., a Pol III promoter such as U6 or H1 ).
  • a guide RNA can also be precomplexed with a CRISPR-Cas effector protein (i.e., can be used or introduced into a cell as part of an RNP).
  • a guide RNA and/or capNucleicAcid (e.g., capRNA or capDNA) has one or more modifications, e.g., a base modification, a backbone modification, etc., to provide the nucleic acid with a new or enhanced feature (e.g., improved stability).
  • a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to the 2', the 3', or the 5' hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are suitable.
  • linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • 2'-modified or “2'-substituted” means a sugar comprising a substituent at the 2'-position other than H or OH.
  • 2'-modified nucleotides include moieties with 2' substituents selected from alkyl, allyl, amino, azido, fluoro, thio, O- alkyl, e.g., O-methyl, O-allyl, OCF3, O-(CH2)2-O-CH3 (e.g., 2'-O-methoxyethyl (MOE)), O-(CH2)2SCH3, )-(CH2)2-ONR2, and O-CH2C(O)-NR2, where each R is independently selected from H, alkyl, and substituted alkyl.
  • Suitable nucleic acid modifications include, but are not limited to: 2’Omethyl modified nucleotides, 2’ fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and a 5' cap (e.g., a 7-methylguanylate cap (m7G)). Additional details and additional modifications are described below.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • a 2'-O-Methyl modified nucleotide (also referred to as 2'-O-Methyl RNA) is a naturally occurring modification of RNA found in tRNA and other small RNAs that arises as a post-transcriptional modification. Oligonucleotides can be directly synthesized that contain 2'-O-Methyl RNA. This modification increases Tm of RNA:RNA duplexes but results in only small changes in RNA:DNA stability. It is stabile with respect to attack by single-stranded ribonucleases and is typically 5 to 10-fold less susceptible to DNases than DNA. It is commonly used in antisense oligos as a means to increase stability and binding affinity to the target message.
  • Fluoro modified nucleotides e.g., 2' Fluoro bases
  • 2' Fluoro bases have a fluorine modified ribose which increases binding affinity (Tm) and also confers some relative nuclease resistance when compared to native RNA. These modifications are commonly employed in ribozymes and siRNAs to improve stability in serum or other biological fluids.
  • LNA bases have a modification to the ribose backbone that locks the base in the C3'-endo position, which favors RNA A-type helix duplex geometry. This modification significantly increases Tm and is also very nuclease resistant. Multiple LNA insertions can be placed in an oligo at any position except the 3'-end.
  • the phosphorothioate (PS) bond (i.e., a phosphorothioate linkage) substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of a nucleic acid (e.g., an oligo). This modification renders the internucleotide linkage resistant to nuclease degradation.
  • Phosphorothioate bonds can be introduced between the last 3-5 nucleotides at the 5'- or 3'-end of the oligo to inhibit exonuclease degradation. Including phosphorothioate bonds within the oligo (e.g., throughout the entire oligo) can help reduce attack by endonucleases as well.
  • a guide RNA and/or capNucleicAcid (e.g., capRNA or capDNA) has one or more nucleotides that are 2'-O-Methyl modified nucleotides.
  • a guide RNA and/or capNucleicAcid (e.g., capRNA or capDNA) has one or more 2’ Fluoro modified nucleotides.
  • a guide RNA and/or capNucleicAcid e.g., capRNA or capDNA
  • a guide RNA and/or capNucleicAcid (e.g., capRNA or capDNA) has one or more nucleotides that are linked by a phosphorothioate bond (i.e., the guide RNA and/or capNucleicAcid (e.g., capRNA or capDNA) has one or more phosphorothioate linkages).
  • guide RNA and/or capNucleicAcid (e.g., capRNA or capDNA) has a 5’ cap (e.g., a 7-methylguanylate cap (m7G)).
  • a guide RNA and/or capNucleicAcid has a combination of modified nucleotides.
  • a guide RNA and/or capNucleicAcid e.g., capRNA or capDNA
  • can have a 5’ cap e.g., a 7-methylguanylate cap (m7G)
  • m7G 7-methylguanylate cap
  • a 2'-O- Methyl nucleotide and/or a 2’ fluoro modified nucleotide and/or a LNA base and/or a phosphorothioate linkage e.g., a 2'-O- Methyl nucleotide and/or a 2’ fluoro modified nucleotide and/or a LNA base and/or a phosphorothioate linkage.
  • Examples of suitable guide RNA and/or capNucleicAcid (e.g., capRNA or capDNA)s containing modifications include guide RNA and/or capNucleicAcid (e.g., capRNA or capDNA)s with modified backbones or non-natural internucleoside linkages.
  • Guide RNA and/or capNucleicAcid (e.g., capRNA or capDNA) having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • Suitable modified nucleic acid backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or
  • Suitable oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts such as, for example, potassium or sodium), mixed salts and free acid forms are also included.
  • Nucleoside subunits can be joined by a variety of intersubunit linkages, including, but not limited to, phosphodiester, phosphotriester, an alkylphosphonate, e.g., methylphosphonate, P3' ⁇ N5' phosphoramidate, N3' >P5' phosphoramidate, N3' >P5' thiophosphoramidate, phosphorodiamidate, and phosphorothioate linkages.
  • intersubunit linkage has a chiral atom.
  • Representative chiral intersubunit linkages include, but are not limited to, alkylphosphonates, phosphorodiamidates and phosphorothioates.
  • oligonucleotides includes chemical and biochemical modifications, such as those known to one skilled in the art, e.g., to the sugar (e.g., 2' substitutions), the base (see the definition of “nucleoside” below), and/or the 3’ and 5' termini.
  • each linkage may be formed using the same chemistry or a mixture of linkage chemistries may be used.
  • one or more of the linkages may be chiral. Linkages having a chiral atom can be prepared as racemic mixtures, or as separate enantiomers.
  • MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677, the disclosure of which is incorporated herein by reference in its entirety.
  • Suitable amide internucleoside linkages are disclosed in U.S. Pat. No. 5,602,240, the disclosure of which is incorporated herein by reference in its entirety.
  • nucleic acids having morpholino backbone structures as described in, e.g., U.S. Pat. No. 5,034,506.
  • a subject nucleic acid comprises a 6-membered morpholino ring in place of a ribose ring.
  • a phosphorodiamidate or other non- phosphodiester internucleoside linkage replaces a phosphodiester linkage.
  • Suitable modified polynucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • riboacetyl backbones alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
  • a subject guide RNA and/or capNucleicAcid can include a nucleic acid mimetic.
  • the term "mimetic" as it is applied to polynucleotides is intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate.
  • the heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid.
  • PNA peptide nucleic acid
  • the sugar-backbone of a polynucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleotides are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • PNA peptide nucleic acid
  • the backbone in PNA compounds is two or more linked aminoethylglycine units which gives PNA an amide containing backbone.
  • the heterocyclic base moieties are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative U.S. patents that describe the preparation of PNA compounds include, but are not limited to: U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262, the disclosures of which are incorporated herein by reference in their entirety.
  • Another class of polynucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring.
  • a number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid.
  • One class of linking groups has been selected to give a non-ionic oligomeric compound.
  • the non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins.
  • Morpholino-based polynucleotides are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A.
  • Morpholino-based polynucleotides are disclosed in U.S. Pat. No. 5,034,506, the disclosure of which is incorporated herein by reference in its entirety. A variety of compounds within the morpholino class of polynucleotides have been prepared, having a variety of different linking groups joining the monomeric subunits.
  • CeNA cyclohexenyl nucleic acids
  • the furanose ring normally present in a DNA/RNA molecule is replaced with a cyclohexenyl ring.
  • CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry.
  • Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602, the disclosure of which is incorporated herein by reference in its entirety).
  • CeNA monomers In general the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation.
  • a further modification includes Locked Nucleic Acids (LNAs) in which the 2'- hydroxyl group is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C,4'-C-oxymethylene linkage thereby forming a bicyclic sugar moiety.
  • the linkage can be a methylene (-CH2-), group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456, the disclosure of which is incorporated herein by reference in its entirety).
  • Potent and nontoxic antisense oligonucleotides containing LNAs have been described (e.g., Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638, the disclosure of which is incorporated herein by reference in its entirety).
  • LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226, as well as U.S. applications 20120165514, 20100216983, 20090041809, 20060117410, 20040014959, 20020094555, and 20020086998, the disclosures of which are incorporated herein by reference in their entirety.
  • a “bicyclic nucleic acid” or a “bridged nucleic acid” refers to a modified RNA nucleotide where the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon, thereby forming a bicyclic ring system.
  • BNA monomers can contain a five-membered, six-membered or a seven-membered bridge structure with a fixed 3'-endo conformation.
  • Bridged nucleic acids include without limitation, locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA) and constrained ethyl (cEt).
  • a “bridge” refers to a chain of atoms or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of a ring system (e.g., the ribose ring system) which is bonded to three or more skeletal atoms (excluding hydrogen).
  • the bridge in a BNA has 7-12 ring members and 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • a BNA is optionally substituted with one or more substituents, e.g., including, but not limited to alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, amino and halogen.
  • Ethylene-bridged nucleic acid refers to an LNA modified RNA nucleotide where the ribose moiety is modified with an extra bridge containing two carbon atoms between the 2' oxygen and the 4' carbon (see, e.g., Morita et al., Bioorganic Medicinal Chemistry, 2003, 11 (10), 2211 -2226). Ethylene-bridged nucleic acids are also encompassed by the term “bicyclic nucleic acids” or “bridged nucleic acids” (BN A).
  • a “constrained ethyl (cEt)” refers to an LNA modified RNA nucleotide where the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon, wherein the carbon atom of the bridge includes a methyl group.
  • the cEt is (S)-constrained ethyl.
  • the cEt is (R)-constrained ethyl (see, e.g., Pallan et al., Chem. Commun. (Camb)., 2012, 48(66), 8195-8197).
  • Constrained ethyl nucleic acids are also encompassed by the term “bicyclic nucleic acids” or “bridged nucleic acids” (BNA).
  • a guide RNA and/or capNucleicAcid can also include one or more substituted sugar moieties.
  • Suitable polynucleotides comprise a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.1 to C10 alkyl or C2 to C10 alkenyl and alkynyl.
  • Particularly suitable are O((CH2)nO) mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10.
  • Suitable polynucleotides comprise a sugar substituent group selected from: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a sugar substituent group selected from: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl,
  • a suitable modification includes 2'-methoxyethoxy (2'-O-CH2 CH2OCH3, also known as 2'-O-(2-methoxyethyl) (or 2'-MOE or 2’-O-MOE-RNA) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504, the disclosure of which is incorporated herein by reference in its entirety) i.e., an alkoxyalkoxy group.
  • a further suitable modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'- dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy- ethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2.
  • 2'-dimethylaminooxyethoxy i.e., a O(CH2)2ON(CH3)2 group
  • 2'-DMAOE also known as 2'-DMAOE
  • 2'- dimethylaminoethoxyethoxy also known in the art as 2'-O-dimethyl-amino-ethoxy- ethyl or 2'-DMAEOE
  • 2'-sugar substituent groups may be in the arabino (up) position or ribo (down) position.
  • a suitable 2'-arabino modification is 2'-F.
  • Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • a guide RNA and/or capNucleicAcid may also include nucleobase (often referred to in the art simply as “base' 1 ) modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidi ne( 1 H- pyrimido(5,4-b)(1 ,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1 H- pyrimido(5,4-b)(1 ,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
  • Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.
  • nucleobases are useful for increasing the binding affinity of an oligomeric compound.
  • These include 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1 .2° C.
  • RNA and/or capNucleicAcid e.g., capRNA or capDNA
  • moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups.
  • Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Suitable conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of a subject nucleic acid.
  • Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553- 6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053- 1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem.
  • lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553- 6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 10
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
  • CRISPR-Cas effector proteins can be from Class 1 or Class 2 CRISPR systems.
  • the CRISPR-Cas effector protein of Class 1 CRISPR systems is a multi-subunit complex of proteins (e.g., Cas10/Csm1 , Csm2, Csm3, Csm4, and Csm5), while the CRISPR-Cas effector protein of Class 2 CRISPR systems is a single protein that carries out the effector functions.
  • a CRISPR-Cas effector protein is a Class 1 CRISPR-Cas effector protein, e.g., a Type I, Type III, or Type IV CRISPR-Cas effector protein.
  • a CRISPR-Cas effector protein is a Class 2 CRISPR-Cas effector protein, e.g., a Type II, Type V, or Type VI CRISPR- Cas effector protein. See, e.g., Zetsche et al, Cell. 2015 Oct 22;163(3):759-71 ; Makarova et al, Nat Rev Microbiol. 2015 Nov;13(11 ):722-36; Shmakov et al., Mol Cell. 2015 Nov 5;60(3):385-97; Shmakov et al., Nat Rev Microbiol.
  • Class 2 CRISPR proteins include, for example, type II CRISPR-Cas proteins (e.g., Cas9), type V CRISPR- Cas proteins (e.g., Cpf1 (Cas12a), C2c1 (Cas12b), C2C3 (Cas12c), CasY (Cas12d), CasX (Cas12e), Cas 12f (also known as Cas14), and type VI CRISPR- Cas proteins (e.g., C2c2 (Cas13a), C2C7 (Cas13c), C2c6 (Cas13b), and the like.
  • type II CRISPR-Cas proteins e.g., Cas9
  • type V CRISPR- Cas proteins e.g., Cpf1 (Cas12a), C2c1 (Cas12b), C2C3 (Cas12c), CasY (Cas12d), CasX (Cas12e
  • Class 2 CRISPR-Cas effector proteins include type II, type V, and type VI CRISPR- Cas proteins, but the term is also meant to encompass any class 2 CRISPR-Cas protein suitable for binding to a corresponding guide RNA and forming a ribonucleoprotein (RNP) complex.
  • RNP ribonucleoprotein
  • CRISPR-Cas effector proteins that exhibit trans-cleavage (also referred to in the art as collateral cleavage) activity.
  • a Cas12 RNP i.e., Cas12 protein complexed with a guide RNA
  • Hybridization between the target and guide RNA activates Cas12, and with the RuvC domain, the active Cas12 cleaves the target (cis- cleavage) and nontarget ssDNA nearby (trans-cleavage).
  • the activated protein promiscuously cleaves single stranded target nucleic acid (e.g., ssDNAs) (i.e., the nuclease cleaves non-target single stranded target nucleic acid, e.g., ssDNAs).
  • a Cas13 RNP i.e., Cas13 protein complexed with a guide RNA
  • trans-cleavage The collateral cleavage of nontarget nucleic acids by active Cas12 and Cas13 systems is generally called trans-cleavage (or collateral cleavage).
  • Cas12 and Cas13 enzymes have been used for killing cells (e.g., when activated inside of cells such as bacterial cells) and for nucleic acid target recognition, signal generation, and amplification.
  • CRISPR-Cas effector proteins with trans-cleavage activity e.g., Cas12, Cas13, and the like
  • CRISPR-Cas effector proteins with trans-cleavage activity can cleave short single-stranded reporter oligos (labeled single stranded detector nucleic acids) through trans-cleavage, e.g., separating the fluorophore from the quencher which are typically labeled at opposite ends of the reporter molecule, and generating measurable fluorescence signals.
  • the technology for CRISPR-based molecular detection is generally based on the specific binding to target DNA or RNA and the trans-cleavage activity against ssRNA or ssDNA that is activated by specific sequence recognition.
  • the target type (RNA or DNA) being detected e.g., in a sample, is not necessarily constant for a particular CRISPR-Cas effector protein.
  • target DNA and target RNA can be interconverted, i.e., converted to the other type (DNA to RNA, or RNA to DNA), through transcription or reverse transcription.
  • the CRISPR-Cas effector protein is a Class 1 protein having transcleavage activity.
  • the CRISPR-Cas effector protein is Type III protein (e.g., a Csm (Type 11 IA) complex). In some cases, the CRISPR-Cas effector protein is a Csm (Type 111 A) complex. In some cases, the CRISPR-Cas effector protein is a Cmr (Type II IB) complex.
  • the CRISPR-Cas effector protein is Class 2 protein having transcleavage activity.
  • the CRISPR-Cas effector protein is Type VI protein (e.g., a Cas13 protein).
  • the CRISPR-Cas effector protein is a Cas13 protein.
  • the CRISPR-Cas effector protein is a Cas13a protein.
  • the CRISPR-Cas effector protein is a Cas13b protein.
  • the CRISPR-Cas effector protein is a Cas13d protein.
  • the CRISPR-Cas effector protein is Type V protein (e.g., a Cas12 protein).
  • the CRISPR-Cas effector protein is a Cas12 protein. In some cases, the CRISPR-Cas effector protein is a Cas12a protein. In some cases, the CRISPR-Cas effector protein is a Cas12b protein. In some cases, the CRISPR-Cas effector protein is a Cas12c protein. In some cases, the CRISPR-Cas effector protein is a Cas12c1 protein. In some cases, the CRISPR-Cas effector protein is a Cas12d protein. In some cases, the CRISPR-Cas effector protein is a Cas12f (also known as Cas14 or Cas14a) protein.
  • the CRISPR-Cas effector protein is a Cas12i protein. In some cases, the CRISPR-Cas effector protein is a Cas12i2 protein. See, e.g., Huang et al., Biosensors (Basel). 2022 Sep 20;12(10):779; Feng et al., Anal Chem. 2023 Jan 10;95(1 ):206-217; He et al., Genes 2023, 14, 850; and Li et al., Front Mol Biosci. 2023 Sep 21 ;10:1260883.
  • CRISPR-Cas effector proteins are known in the art, e.g., those harboring mutations that increase specificity (e.g., decrease off-targeting), and any convenient variant can be used. See, e.g., Vakulskas et al., Nat Med. 2018 Aug;24(8):1216-1224; Kleinstiver et al., Nature. 2016 Jan 28;529(7587):490-5; Yuen et al., Nucleic Acids Res. 2022 Feb 22;50(3):1650-1660; Wei et al., FASEB J.
  • CRISPR-Cas effector proteins will be readily available to one of ordinary skill in the art, and any convenient CRISPR-Cas effector protein can be used.
  • a subject CRISPR-Cas effector protein will be a Cas12a protein (e.g., Acidaminococcus sp., strain BV3L6 (AsCas12a), LbCas12a, and FnoCas12a). Sequences for these proteins are readily available to one of ordinary skill in the art.
  • Cas12a The amino acid sequence of an example wild-type Cas12a polypeptide (Acidaminococcus sp., strain BV3L6 (AsCas12a)) is provided as SEQ ID NO: 234.
  • Other examples of Cas12a include, but are not limited to: LbCas12a and FnoCas12a, as well as AsCas12a ultra nuclease (see, e.g., Zhang et al., Nat Commun. 2021 Jun 23;12(1 ):3908).
  • Examples of naturally existing Cas12a proteins are set forth as SEQ ID NOs: 233- 245.
  • a subject Cas12 protein includes an amino acid sequence having 80% or more (e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100%) amino acid sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 233-245.
  • a suitable Cas12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Cas12a amino acid sequence set forth in SEQ ID NO: 234.
  • a Cas13 effector protein has two HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domains that provide RNase activity.
  • the Cas13 protein when associated with crRNA, forms an RNA-guided RNA targeting complex to recognize and cleave ssRNA targets.
  • this system is further classified into six subtypes: Vl-A (Cas13a, C2c2), Vl-B (Cas13b, C2c4), Vl-C (Cas13c, C2c7), Vl-D (Cas13d), Vl-X (Cas13X) and Vl-Y (Cas13Y). All Cas13 proteins possess two enzymatically distinct RNase activities, which include processing pre-crRNA into mature functional crRNA and the degradation of target RNA by the HEPN domains. The location of these HEPN domains differs based on the type of Cas13 proteins.
  • the HEPN domains are present at the center and C terminus, whereas in Cas13b, Cas13X, and Y, they are located at the N-terminus and C-terminus of the proteins.
  • the HEPN domains of Cas13 proteins can cleave not only the desired target, but also exhibit a non-specific collateral cleavage activity resulting in the degradation of the RNA near the Cas13 complex.
  • the length of the crRNA or the spacer sequence varies (e.g., 24-30 nt) with the type of Cas13.
  • Cas13a has many orthologs such as Listeria seeligeri (Lse) and Leptotrichia wadei (Lwa), Leptotrichia buccalis (Lbu), and Lachnospiraceae bacterium (Lba).
  • Wild type Cas13a CRISPR arrays typically consists of a 5' 28 nt direct repeat (DR) unique to each ortholog and a 28-30 nt spacer sequence (complementary to the target sequence). As such, for a Cas13a guide RNA, the constant region is 5’ of the guide sequence.
  • LshCas13a have a 3' H (non-G), a single base protospacer flanking site (PFS) preference, whereas LwaCas13a and LbuCas13a do not show any PFS preference.
  • Cas13b has its direct repeat (DR) on the 3' end of crRNA compared to the 5' DR present in Cas13a, Cas13c, and Cas13d.
  • Cas13b orthologs such as Bergeyella zoohelcum (BzCas13b) and Porphyromonas gulae (PguCas13b) prefer 5' PFS of D (A, U, or G) and 3' PFS of NAN or NNA.
  • PspCas13b has no PFS requirement.
  • Cas13b is further differentiated into two types based on the presence of regulatory accessory proteins csx27 and csx28 that can repress or enhance the RNA interference activity of Cas13b, respectively.
  • Cas13c has its direct repeat (DR) on the 5' end of crRNA and a spacer length (e.g., 28-30 nt), similar to that of Cas13a and Cas13d.
  • Cas13c orthologs include Fusobacterium perfoetens (FpeCas13c).
  • Cas13d is the smallest of Cas13a-d.
  • the Cas13d from the Ruminococcus flavefaciens XPD3002 (CasRx/RfxCas13d) is a prominent homolog for multiple organisms.
  • Type Vl-D has no PFS constraints like the other Cas13 enzymes.
  • Example naturally existing Cas13a proteins are set forth as SEQ ID NOs: 1 -66 (see Table 1 ).
  • a subject Cas13 protein includes an amino acid sequence having 80% or more (e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100%) amino acid sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 -66 .
  • a suitable Cas13 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Leptotrichia buccalis Cas13a amino acid sequence set forth in SEQ ID NO: 1 .
  • Example naturally existing Cas13b proteins are set forth as SEQ ID NOs: 67-200 (see Table 1 ).
  • a subject Cas13b protein includes an amino acid sequence having 80% or more (e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100%) amino acid sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 67-200.
  • Example naturally existing Cas13c proteins are set forth as SEQ ID NOs: 201 -212.
  • a subject Cas13c protein includes an amino acid sequence having 80% or more (e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100%) amino acid sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 201 -212.
  • Example naturally existing Cas13d proteins are set forth as SEQ ID NOs: 213-224.
  • a subject Cas13d protein includes an amino acid sequence having 80% or more (e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100%) amino acid sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 213-224.
  • the CRISPR-Cas effector protein is a Class 1 protein (i.e., a multisubunit protein).
  • the CRISPR-Cas effector protein is a Type III CRISPR-Cas effector polypeptide.
  • the CRISPR-Cas effector polypeptide is a multi-subunit Type 111 A CRISPR-Cas effector polypeptide comprising Cas10/Csm1 , Csm2, Csm3, Csm4, and Csm5 polypeptides.
  • the natural multiprotein Csm complex comprises the five subunits (Csm1 -5) in varying stoichiometries and relies on an additional protein, Cas6, for processing the precursor crRNA.
  • the CRISPR-Cas effector polypeptide is a multisubunit Type 11 IB CRISPR-Cas effector polypeptide comprising Cmr1 , Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6 subunits.
  • the Class 1 CRISPR system will be a Type I CRISPR-Cas effector polypeptide comprising Cas7.
  • the present disclosure provides methods of activating a CRISPR-Cas effector protein with a nucleic acid of interest (actNucleicAcid).
  • methods generally include contacting a capNucleicAcid (described herein) with a CRISPR-Cas effector protein (described herein) and a CRISPR-Cas guide RNA (described herein) in the presence of a nucleic acid of interest (actNucleicAcid) (described herein).
  • a method is a method of detecting a nucleic acid of interest in a sample. Such a method usually takes place outside of a cell (i.e. , not inside of a cell).
  • the contacting takes place inside of a cell that includes the actNucleicAcid.
  • Such methods can be used, e.g., as a way to kill cells, but only those cells that include actNucleicAcid.
  • introduction of components of a split guide RNA system as disclosed herein can be used to specifically target the cancer cells for killing.
  • the activated transcleavage activity of the CRISPR-Cas effector protein e.g., Cas13a, Cas12a, and the like
  • the split guide system can be used to target prokaryotic cells (those expressing a particular actNucleicAcid) for death.
  • RNAs and/or DNAs RNAs and/or DNAs
  • proteins including preformed RNPs
  • any convenient technique can be used, e.g., lipofection, nucleofection, viral transduction, electroporation, injection, etc.
  • a nucleic acid of interest can be any RNA (e.g., single-stranded RNA or double-stranded RNA) or any DNA (e.g., single-stranded DNA or double-stranded DNA), as long as the targeted sequence hybridize with the capture region of the capNucleicAcid (e.g., capRNA, capDNA).
  • the actNucleicAcid is single stranded.
  • the actNucleicAcid is an RNA.
  • the actNucleicAcid is a DNA.
  • a Cas13 protein e.g., Cas13a
  • the capNucleicAcid is a single stranded RNA
  • the actNucleicAcid is also a single stranded RNA (e.g., a miRNA).
  • a Cas12 protein e.g., Cas12a
  • the capNucleicAcid is a single stranded DNA
  • the actNucleicAcid is a single stranded RNA (e.g., a miRNA).
  • a Cas12 protein e.g., Cas12a
  • the capNucleicAcid is a single stranded DNA
  • the actNucleicAcid is a single stranded DNA.
  • a Cas12 protein e.g., Cas12a
  • the capNucleicAcid is a double stranded DNA
  • the actNucleicAcid is a single stranded RNA (e.g., a miRNA).
  • a Cas12 protein e.g., Cas12a
  • the capNucleicAcid is a double stranded DNA
  • the actNucleicAcid is a single stranded DNA.
  • the targeted sequence of an actNucleicAcid (e.g., actRNA or actDNA) (i.e., the sequence that hybridizes with the capture region of a capNucleicAcid) is 8- 26 nucleotides (nt) long (e.g., 8-24, 8-23, 8-22, 8-20, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11 , 8-10, 9-26, 9-24, 9-23, 9-22, 9-20, 9-18, 9-17, 9-16, 9-15, 9-14, 9- 13, 9-12, 9-11 , 9-10, 10-26, 10-24, 10-23, 10-22, 10-20, 10-18, 10-17, 10-16, IQ- 15, 10-14, 10-13, 10-12, 10-11 , 12-26, 12-24, 12-23, 12-22, 12-20, 12-18, 12-17, 12-16, 12-15, 12-14, or 12-13 nt).
  • the targeted sequence of the actNucleicAcid is 8- 26 nucleot
  • the actNucleicAcid is 30-5000 nt long (e.g., 30-3000, 30-2500, 30-2400, 30-2000, 30-1500, 30-1000, 30-800, 30-500, 30-250, 50-3000, 50-2500, 50-2400, 50-2000, 50-1500, 50-1000, 50-800, 50-500, 50-250, 100-3000, 100- 2500, 100-2400, 100-2000, 100-1500, 100-1000, 100-800, 100-500, 100-250, 250- 3000, 250-2500, 250-2400, 250-2000, 250-1500, 250-1000, 250-800, or 250-500 nt).
  • the actNucleicAcid is 100-3000 nt long (e.g., 100-2500, 100- 2400, 100-2000, 100-1500, 100-1000, 100-800, 100-500, 100-250, 250-2500, 250- 2400, 250-2000, 250-1500, 250-1000, 250-800, or 250-500 nt).
  • the actNucleicAcid is -2500 nt long.
  • the actNucleicAcid is greater than 1000 nt long. In some cases, the actNucleicAcid is greater than 2000 nt long.
  • the actNucleicAcid is longer than the targeted sequence (e.g., longer than 26 nucleotides).
  • the targeted sequence (the portion of the actNucleicAcid that will hybridize with the capture region of the capNucleicAcid) is at the 5' end. See, e.g, FIG. 14.
  • the sequence detected by the split guide RNA system is part of a longer nucleic acid.
  • the actNucleicAcid is longer than 26 nt, and the targeted sequence (e.g., 8-26 nt) within the actNucleicAcid that acts with the split guide RNA to hybridize to the capNucleicAcid is at the 5’ end of the actNucleicAcid.
  • the targeted sequence e.g. 8-26 nt
  • FIG. 14 illustrates the detection of a sequence in the non coding 5’ UTR region of a viral RNA, where the targeted sequence is shared among different viral RNAs - the presence of any one of the RNAs will be detected.
  • the 8-26 5’ most nucleotides of the actNucleicAcid hybridize to the capture region of the capNucleicAcid. In some embodiments, the 8-16 5’ most nucleotides of the actNucleicAcid hybridize to the capture region of the capNucleicAcid.
  • the actNucleicAcid (e.g., actRNA) is 8-26 nucleotides (nt) long (e.g., 8-24, 8-23, 8-22, 8-20, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11 , 8-10, 9- 26, 9-24, 9-23, 9-22, 9-20, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11 , 9-10, IQ- 26, 10-24, 10-23, 10-22, 10-20, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11 , 12-26, 12-24, 12-23, 12-22, 12-20, 12-18, 12-17, 12-16, 12-15, 12-14, or 12- 13 nt).
  • actRNA is 8-26 nucleotides (nt) long (e.g., 8-24, 8-23, 8-22, 8-20, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8
  • the actNucleicAcid e.g., actRNA
  • the actNucleicAcid is 8-16 nt long (e.g., 8-15, 8-14, 8-13, 8-12, 8-11 , 8-10, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11 , 9-10, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11 , 12-16, 12-15, 12-14, or 12-13 nt).
  • actRNAs include but are not limited to mRNA, rRNA, tRNA, noncoding RNA (ncRNA), long non-coding RNA (IncRNA), and microRNA (miRNA).
  • the actNucleicAcid is mRNA.
  • the actNucleicAcid is a miRNA (e.g., a human miRNA).
  • the actNucleicAcid is RNA from a virus (e.g., Zika virus, human immunodeficiency virus, influenza virus, and the like).
  • the actNucleicAcid is RNA of a parasite.
  • the actNucleicAcid is RNA of a bacterium, e.g., a pathogenic bacterium.
  • the source of the actNucleicAcid can be the same as the source of a sample (e.g., for methods of detection).
  • detection of a actNucleicAcid, where the actNucleicAcid is an mRNA provides for detection of a DNA encoding the mRNA.
  • the actNucleicAcid is an mRNA present in a diseased cell (e.g., a cancer cell).
  • the actNucleicAcid is a miRNA present in a diseased cell (e.g., a cancer cell).
  • Examples of possible actDNAs include, but are not limited to, viral DNAs such as: a papovavirus (e.g, human papillomavirus (HPV), polyomavirus); a hepadnavirus (e.g., Hepatitis B Virus (HBV)); a herpesvirus (e.g, herpes simplex virus (HSV), varicella zoster virus (VZV), epstein-barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, Pityriasis Rosea, kaposi’s sarcoma-associated herpesvirus); an adenovirus (e.g., atadenovirus, aviadenovirus, ichtadenovirus, mastadenovirus, siadenovirus); a poxvirus (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, orf virus, pseudoco
  • an actNucleicAcid is not subjected to an amplification step.
  • an actNucleicAcid is subject to an amplification step, to generate an amplification product (an amplicon), and the amplification product is detected using a method of the present disclosure.
  • the amplifying comprises recombinase polymerase amplification (RPA), transcription mediated amplification (TMA), strand displacement amplification (SDA), helicase dependent amplification (HDA), loop mediated amplification (LAMP), rolling circle amplification (RCA), single primer isothermal amplification (SPIA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), or improved multiple displacement amplification (IMDA), or nucleic acid sequence-based amplification (NASBA).
  • RPA recombinase polymerase amplification
  • the amplifying comprises loop mediated amplification (LAMP).
  • the source of the actNucleicAcid can be any source.
  • the actNucleicAcid is a viral nucleic acid (e.g., viral RNA, viral DNA, a genomic DNA of a DNA virus, a genomic RNA of an RNA virus).
  • subject method can be for detecting the presence of a viral actNucleicAcid amongst a population of nucleic acids (e.g., in a sample).
  • a subject method can also be used for the cleavage of non-target single stranded nucleic acids in the present of an actNucleicAcid.
  • a subject method can be used to promiscuously cleave non-target single stranded nucleic acids in the cell (single stranded nucleic acids that do not hybridize with the guide sequence of the guide RNA) when a particular actNucleicAcid is present in the cell (e.g., when the cell is infected with a virus and viral target nucleic acid is detected).
  • Detection e.g., when the cell is infected with a virus and viral target nucleic acid is detected.
  • the components described herein are used in a method of detection - to detect the presence of a nucleic acid of interested (an actNucleicAcid).
  • an actNucleicAcid e.g., an RNA (actRNA) or a DNA (actDNA) in a sample.
  • the sample is a cell-free sample.
  • the sample comprises cells.
  • the sample comprises a cell lysate.
  • the methods include contacting a sample (e.g., a sample having a plurality of nucleic acids, e.g., RNAs) with (a) a CRISPR-Cas effector protein (e.g., a Cas13 such as Cas13a); (b) a CRISPR-Cas guide RNA (e.g., a Cas13 guide RNA) that hybridizes with the anchor region of a capture nucleic acid (capNucleicAcid); (c) the capture nucleic acid (capNucleicAcid) (e.g., capRNA) - which has an anchor region that hybridizes with the guide RNA and a capture region that hybridizes with the actNucleicAcid (e.g., actRNA) if the actNucleicAcid is present in the sample; and (d) a labeled single stranded detector nucleic acid (which does not hybridize with the CRISPR-Cas guide RNA).
  • a sample e
  • the trans cleavage activity of the CRISPR-Cas effector protein is activated if the actNucleicAcid is present in the sample being contacted.
  • a subject CRISPR- Cas effector protein e.g., Cas13 such as Cas13a
  • Cas13 such as Cas13a
  • RNP ribonucleoprotein complex
  • the CRISPR-Cas effector protein is activated and functions as an endoribonuclease that non-specifically cleaves RNAs (including non-target RNAs) present in the sample.
  • the actNucleicAcid when the actNucleicAcid is present in the sample (e.g., in some cases above a threshold amount), the result is cleavage of single stranded (ss) nucleic acids (e.g., ssRNA and/or ssDNA) (including non-actNucleicAcid) in the sample, which can be detected using any convenient detection method (e.g., using a labeled detector nucleic acid (e.g., detector RNA or DNA)).
  • the contacting step is generally carried out in a composition comprising divalent metal ions.
  • the contacting step can be carried out in an acellular environment, e.g., outside of a cell.
  • the contacting step can be carried out inside a cell.
  • the contacting step can be carried out in a cell in vitro.
  • the contacting step can be carried out in a cell ex vivo.
  • the contacting step can be carried out in a cell in vivo.
  • the guide RNA e.g., Cas13 guide RNA
  • the CRISPR-Cas effector protein e.g., Cas13a, Cas12a, and the like
  • the Guide RNA is provided as DNA encoding the guide RNA
  • the CRISPR-Cas effector protein e.g., Cas13, Cas12, and the like
  • the Guide RNA is provided as RNA; and the CRISPR-Cas effector protein (e.g., Cas13, Cas12, and the like) is provided as RNA encoding the CRISPR-Cas effector protein (e.g., Cas13, Cast 2, and the like).
  • the Guide RNA is provided as DNA encoding the guide RNA; and CRISPR-Cas effector protein (e.g., Cas13, Cas12, and the like) is provided as RNA encoding the CRISPR-Cas effector protein (e.g., Cas13, Cas12, and the like).
  • the Guide RNA is provided as RNA; and the CRISPR-Cas effector protein (e.g., Cas13, Cas12, and the like) is provided as DNA comprising a nucleotide sequence encoding the CRISPR-Cas effector protein (e.g., Cas13, Cas12, and the like).
  • the Guide RNA is provided as DNA encoding the guide RNA; and the CRISPR-Cas effector protein (e.g., Cas13, Cas12, and the like) is provided as DNA comprising a nucleotide sequence encoding the CRISPR-Cas effector protein (e.g., Cas13, Cas12, and the like).
  • the method detects a control or standard nucleic acid (e.g, RNA). The detection of the control provides an internal control that indicates that the method is working.
  • the sample is contacted for 2 hours or less (e.g., 1 .5 hours or less, 1 hour or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, or 5 minutes or less, or 1 minute or less) prior to the measuring step.
  • the sample is contacted for 40 minutes or less prior to the measuring step.
  • the sample is contacted for 20 minutes or less prior to the measuring step.
  • the sample is contacted for 10 minutes or less prior to the measuring step.
  • the sample is contacted for 5 minutes or less prior to the measuring step. In some cases, the sample is contacted for 1 minute or less prior to the measuring step. In some cases, the sample is contacted for from 50 seconds to 60 seconds prior to the measuring step. In some cases, the sample is contacted for from 40 seconds to 50 seconds prior to the measuring step. In some cases, the sample is contacted for from 30 seconds to 40 seconds prior to the measuring step. In some cases, the sample is contacted for from 20 seconds to 30 seconds prior to the measuring step. In some cases, the sample is contacted for from 10 seconds to 20 seconds prior to the measuring step.
  • the includes a plurality of nucleic acids (e.g., RNAs or DNAs) (e.g., comprising a actNucleicAcid and a plurality of non-actNucleicAcids).
  • a plurality of nucleic acids e.g., RNAs or DNAs
  • Detecting an actNucleicAcid e.g., a single-stranded actNucleicAcid such as RNA
  • a sample comprising a plurality of nucleic acids (e.g., RNAs or DNAs) (including the actNucleicAcid and a plurality of non-actNucleicAcids) can detect a actNucleicAcid with a high degree of sensitivity.
  • the actNucleicAcid is present at one or more copies per 10 7 non-actNucleicAcids (e.g., one or more copies per 10® non-actNucleicAcids, one or more copies per 10 5 non-actNucleicAcids, one or more copies per 10 4 non-actNucleicAcids, one or more copies per 10 3 non- actNucleicAcids, one or more copies per 10 2 non-actNucleicAcids, one or more copies per 50 non-actNucleicAcids, one or more copies per 20 non- actNucleicAcids, one or more copies per 10 non-actNucleicAcids, or one or more copies per 5 non-actNucleicAcids).
  • non-actNucleicAcids e.g., one or more copies per 10® non-actNucleicAcids, one or more copies per 10 5 non-actNucleicAcids, one or more copies per 10 4 non-actNucleicAcids, one or more copies
  • a method of the present disclosure can detect a actNucleicAcid present in a sample comprising a plurality of nucleic acids (e.g., RNAs or DNAs) (including the actNucleicAcid and a plurality of non-actNucleicAcids), where the actNucleicAcid is present at from one copy per 10 7 non-actNucleicAcids to one copy per 10 non-actNucleicAcids (e.g., from 1 copy per 10 7 non-actNucleicAcids to 1 copy per 10 2 non-actNucleicAcids, from 1 copy per 10 7 non-actNucleicAcids to 1 copy per 10 3 non-actNucleicAcids, from 1 copy per 10 7 non-actNucleicAcids to 1 copy per 10 4 non-actNucleicAcids, from 1 copy per 10 7 non-actNucleicAcids to 1 copy per 10 5 non-actNucleicAcids, from 1 copy per 10 7
  • nucleic acids
  • a method of the present disclosure can detect a actNucleicAcid present in a sample comprising a plurality of nucleic acids (e.g., RNAs or DNAs) (including the actNucleicAcid and a plurality of non-actNucleicAcids), where the target single-stranded RNA is present at from one copy per 10 7 non- actNucleicAcids to one copy per 100 non-actNucleicAcids (e.g., from 1 copy per
  • the threshold of detection for a subject method of detecting a actNucleicAcid in a sample, is 1 pM or less.
  • the term “threshold of detection” is used herein to describe the minimal amount of actNucleicAcid that must be present in a sample in order for detection to occur.
  • a threshold of detection when a threshold of detection is 1 pM, then a signal can be detected when a actNucleicAcid is present in the sample at a concentration of 1 pM or more.
  • a method of the present disclosure has a threshold of detection of 500 fM or less. In some cases, a method of the present disclosure has a threshold of detection of 200 fM or less.
  • a method of the present disclosure has a threshold of detection of 100 fM or less. In some cases, a method of the present disclosure has a threshold of detection of 10 fM or less. In some cases, a method of the present disclosure has a threshold of detection of 1 fM or less. In some cases, a method of the present disclosure has a threshold of detection of 500 aM or less. In some cases, a method of the present disclosure has a threshold of detection of 250 aM or less. In some cases, a method of the present disclosure has a threshold of detection of 100 aM or less. In some cases, a method of the present disclosure has a threshold of detection of 10 aM or less.
  • a method of the present disclosure has a threshold of detection of 250 fM or less. In some cases, a method of the present disclosure has a threshold of detection of 50 fM or less. In some cases, a method of the present disclosure has a threshold of detection of about 10 fM. In some cases, a method of the present disclosure has a threshold of detection of about 50 fM. In some cases, a method of the present disclosure has a threshold of detection of about 100 fM. In some cases, a method of the present disclosure has a threshold of detection of about 150 fM. In some cases, a method of the present disclosure has a threshold of detection of about 200 fM.
  • the threshold of detection (for detecting the actNucleicAcid in a subject method), is in a range of from 1 aM to 1 nM (e.g., from 1 aM to 500 fM, from 10 aM to 500 fM, from 50 aM to 500 fM, from 250 aM to 500 fM, 500 aM to 500 fM, from 1 aM to 300 fM, from 10 aM to 300 fM, from 50 aM to 300 fM, from 250 aM to 300 fM, 500 aM to 300 fM, from 100 fM to 500 pM, from 100 fM to 200 pM, from 100 fM to 100 pM, from 100 fM to 10 pM, from 100 fM to 1 pM, from 100 fM to 750 fM, from 200 fM to 1 nM, from 200 fM to 500 pM, from 200 fM to 200
  • the threshold of detection (for detecting the actNucleicAcid in a subject method), is in a range of from 100 fM to 1 nM (e.g., from 100 fM to 500 pM, from 100 fM to 200 pM, from 100 fM to 100 pM, from 100 fM to 10 pM, from 100 fM to 1 pM, from 100 fM to 750 fM, from 200 fM to 1 nM, from 200 fM to 500 pM, from 200 fM to 200 pM, from 200 fM to 100 pM, from 200 fM to 10 pM, from 200 fM to 1 pM, from 200 fM to 750 fM, from 500 fM to 1 nM, from 500 fM to 500 pM, from 500 fM to 200 pM, from 500 fM to 200 pM, from 500 fM to 100 pM, from 500 fM to 10 pM,
  • a method of the present disclosure has a threshold of detection in a range of from 100 fM to 100 pM. In some cases, a method of the present disclosure has a threshold of detection in a range of from 100 fM to 750 fM. In some cases, a method of the present disclosure has a threshold of detection in a range of from 10 fM to 500 fM, e.g., from 10 fM to 50 fM, from 50 fM to 100 fM, from 100 fM to 250 fM, or from 250 fM to 500 fM. In some cases, a method of the present disclosure has a threshold of detection about 50 fM.
  • a method of the present disclosure has a threshold of detection about 100 fM. In some cases, a method of the present disclosure has a threshold of detection about 150 fM. In some cases, a method of the present disclosure has a threshold of detection about 200 fM.
  • the minimum concentration at which a actNucleicAcid can be detected in a sample is in a range of from 1 aM to 1 nM (e.g., from 1 aM to 500 fM, from 10 aM to 500 fM, from 50 aM to 500 fM, from 250 aM to 500 fM, 500 aM to 500 fM, from 1 aM to 300 fM, from 10 aM to 300 fM, from 50 aM to 300 fM, from 250 aM to 300 fM, 500 aM to 300 fM, from 100 fM to 500 pM, from 100 fM to 200 pM, from 100 fM to 100 pM, from 100 fM to 10 pM, from 100 fM to 1 pM, from 100 fM to 750 fM, from 200 fM to 1 nM, from 200 fM to 500 pM, from 200 fM to 200 .
  • the minimum concentration at which a actNucleicAcid can be detected in a sample is in a range of from 100 fM to 1 nM (e.g., from 100 fM to 500 pM, from 100 fM to 200 pM, from 100 fM to 100 pM, from 100 fM to 10 pM, from 100 fM to 1 pM, from 100 fM to 750 fM, from 200 fM to 1 nM, from 200 fM to 500 pM, from 200 fM to 200 pM, from 200 fM to 100 pM, from 200 fM to 10 pM, from 200 fM to 1 pM, from 200 fM to 750 fM, from 500 fM to 1 nM, from 500 fM to 500 pM, from 500 fM to 200 pM, from 500 fM to 100 pM, from 500 fM to 10 pM, from 500 fM to 100 pM, from
  • the minimum concentration at which a single stranded actNucleicAcid can be detected in a sample is in a range of from 100 fM to 100 pM. In some cases, the minimum concentration at which a single stranded actNucleicAcid can be detected in a sample is in a range of from 100 fM to 750 fM. In some cases, the minimum concentration at which a single stranded actNucleicAcid can be detected in a sample is in a range of from 10 fM to 500 fM, e.g., from 10 fM to 50 fM, from 50 fM to 100 fM, from 100 fM to 250 fM, or from 250 fM to 500 fM.
  • the minimum concentration at which a single stranded actNucleicAcid can be detected in a sample is about 50 fM. In some cases, the minimum concentration at which a single stranded actNucleicAcid can be detected in a sample is about 100 fM. In some cases, the minimum concentration at which a single stranded actNucleicAcid can be detected in a sample is about 150 fM. In some cases, the minimum concentration at which a single stranded actNucleicAcid can be detected in a sample is about 200 fM. In some cases, the minimum concentration at which a single stranded actNucleicAcid can be detected in a sample is about 1 aM.
  • the minimum concentration at which a single stranded actNucleicAcid can be detected in a sample is about 50 aM. In some cases, the minimum concentration at which a single stranded actNucleicAcid can be detected in a sample is about 250 aM. In some cases, the minimum concentration at which a single stranded actNucleicAcid can be detected in a sample is about 500 aM.
  • a method of the present disclosure can detect a actNucleicAcid present in a sample comprising a plurality of actNucleicAcids (including the actNucleicAcid and a plurality of non-actNucleicAcids), where the actNucleicAcid is present at a concentration as low as 1 aM (e.g., as low as 1 aM, as low as 50 aM, as low as 100 aM, as low as 200 aM, as low as 500 aM, as low as 50 fM, as low as 100 fM, as low as 200 fM, as low as 500fM, as low as 800 fM, as low as 1 pM, as low as 10 pM or as low as 100 pM).
  • 1 aM e.g., as low as 1 aM, as low as 50 aM, as low as 100 aM, as low as 200 aM, as low as 500 aM, as low as 50 fM, as low as
  • a method of the present disclosure can be used to determine the amount of a actNucleicAcid in a sample (e.g., a sample comprising the actNucleicAcid and a plurality of non-actNucleicAcids). Determining the amount of a actNucleicAcid in a sample can comprise comparing the amount of detectable signal generated from a test sample to the amount of detectable signal generated from a reference sample.
  • Determining the amount of a actNucleicAcid in a sample can comprise: measuring the detectable signal to generate a test measurement; measuring a detectable signal produced by a reference sample to generate a reference measurement; and comparing the test measurement to the reference measurement to determine an amount of actNucleicAcid present in the sample.
  • a method of the present disclosure for determining the amount of a actNucleicAcid in a sample comprises: a) contacting the sample (e.g., a sample comprising the actNucleicAcid and a plurality of non- actNucleicAcids) with the components described herein (a guide RNA, a capNucleicAcid, and a CRISPR-Cas effector protein), and b) measuring a detectable signal produced by CRISPR-Cas effector protein (e.g., Cas13, Cas12, and the like)-mediated cleavage, generating a test measurement; c) measuring a detectable signal produced by a reference sample to generate a reference measurement; and d) comparing the test measurement to the reference measurement to determine an amount of actNucleicAcid present in the sample.
  • CRISPR-Cas effector protein e.g., Cas13, Cas12, and the like
  • a subject sample can include a plurality of nucleic acids that are not the intended actNucleicAcid.
  • the term “plurality” is used herein to mean two or more.
  • a sample includes two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1 ,000 or more, or 5,000 or more) nucleic acids (e.g., RNAs, DNAs).
  • a subject method can be used as a very sensitive way to detect a single stranded actNucleicAcid (e.g., an RNA) present in a complex mixture of nucleic acids (e.g., RNAs).
  • the sample includes 5 or more nucleic acids (e.g., 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1 ,000 or more, or 5,000 or more RNAs) that differ from one another in sequence.
  • the sample includes 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 10 3 or more, 5 x 10 3 or more, 10 4 or more, 5 x 10 4 or more, 10 5 or more, 5 x 10 5 or more, 10 6 or more 5 x 10 6 or more, or 10 7 or more, nucleic acids that differ from one another in sequence.
  • the sample comprises from 10 to 20, from 20 to 50, from 50 to 100, from 100 to 500, from 500 to 10 3 , from 10 3 to 5 x 10 3 , from 5 x 10 3 to 10 4 , from 10 4 to 5 x 10 4 , from 5 x 10 4 to 10 5 , from 10 5 to 5 x 10 5 , from 5 x 10 5 to 10 6 , from 10 6 to 5 x 10 6 , or from 5 x 10 6 to 10 7 , or more than 10 7 , nucleic acids (e.g. , RNAs) that differ from one another in sequence.
  • nucleic acids e.g. , RNAs
  • the sample comprises from 3 to 10 7 nucleic acids that differ from one another in sequence (e.g., from 5 to 10 6 , from 5 to 10 5 , from 5 to 50,000, from 5 to 30,000, from 10 to 10 6 , from 10 to 10 5 , from 10 to 50,000, from 10 to 30,000, from 20 to 10 6 , from 20 to 10 5 , from 20 to 50,000, or from 20 to 30,000 RNAs that differ from one another in sequence).
  • the sample comprises from 3 to 50,000 nucleic acids that differ from one another in sequence (e.g., from 5 to 30,000, from 10 to 50,000, or from 10 to 30,000) RNAs that differ from one another in sequence).
  • the sample includes 20 or more nucleic acids that differ from one another in sequence.
  • the sample includes nucleic acids from a cell lysate (e.g., a eukaryotic cell lysate, a mammalian cell lysate, a human cell lysate, a prokaryotic cell lysate, a plant cell lysate, and the like).
  • a cell lysate e.g., a eukaryotic cell lysate, a mammalian cell lysate, a human cell lysate, a prokaryotic cell lysate, a plant cell lysate, and the like.
  • the sample includes expressed RNAs from a cell such as a eukaryotic cell, e.g., a mammalian cell such as a human cell.
  • sample is used herein to mean any sample that includes nucleic acids (e.g., single stranded RNAs).
  • the sample can be derived from any source, e.g., the sample can be a synthetic combination of purified nucleic acids; the sample can be a cell lysate, a nucleic acid-enriched cell lysate, or nucleic acids isolated and/or purified from a cell lysate.
  • the sample can be from a patient (e.g., for the purpose of diagnosis).
  • the sample can be from permeabilized cells.
  • the sample can be from crosslinked cells.
  • the sample can be in tissue sections.
  • the sample can be from tissues prepared by crosslinking followed by delipidation and adjustment to make a uniform refractive index.
  • tissue preparation by crosslinking followed by delipidation and adjustment to make a uniform refractive index have been described in, for example, Shah et al., Development (2016) 143, 2862-2867 doi:10.1242/dev.138560.
  • a “sample” can include an actNucleicAcid (e.g., actRNA) and a plurality of nontarget nucleic acids (e.g., DNAs, RNAs).
  • the actNucleicAcid is present in the sample at one copy per 10 non-target nucleic acids, one copy per 20 non-target nucleic acids, one copy per 25 non-target nucleic acids, one copy per 50 non-target nucleic acids, one copy per 100 non-target nucleic acids, one copy per 500 non-target nucleic acids, one copy per 10 3 non-target nucleic acids, one copy per 5 x 10 3 non-target nucleic acids, one copy per 10 4 non-target nucleic acids, one copy per 5 x 10 4 non-target nucleic acids, one copy per 10 5 non-target nucleic acids, one copy per 5 x 10 5 non-target nucleic acids, one copy per 10 6 non-target nucleic acids, or less than one copy per 10 6 non-target nucle
  • the target single-stranded RNA is present in the sample at from one copy per 10 non-target nucleic acids to 1 copy per 20 non-target nucleic acids, from 1 copy per 20 non-target nucleic acids to 1 copy per 50 non-target nucleic acids, from 1 copy per 50 non-target nucleic acids to 1 copy per 100 non-target nucleic acids, from 1 copy per 100 non-target nucleic acids to 1 copy per 500 non-target nucleic acids, from 1 copy per 500 non-target nucleic acids to 1 copy per 10 3 non-target nucleic acids, from 1 copy per 10 3 non-target nucleic acids to 1 copy per 5 x 10 3 non-target nucleic acids, from 1 copy per 5 x 10 3 non-target nucleic acids to 1 copy per 10 4 non-target nucleic acids, from 1 copy per 10 4 non-target nucleic acids to 1 copy per 10 5 non-target nucleic acids, from 1 copy per 10 5 non-target nucleic acids to 1 copy per 10 6 non-target nucleic acids
  • Suitable samples include but are not limited to blood, serum, plasma, urine, aspirate, and biopsy samples.
  • sample with respect to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
  • the definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as cancer cells.
  • the definition also includes sample that have been enriched for particular types of molecules, e.g., RNAs.
  • sample encompasses biological samples such as a clinical sample such as blood, plasma, serum, aspirate, cerebral spinal fluid (CSF), and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, and the like.
  • a “biological sample” includes biological fluids derived therefrom (e.g., cancerous cell, infected cell, etc.), e.g., a sample comprising nucleic acids that is obtained from such cells (e.g., a cell lysate or other cell extract comprising nucleic acids).
  • a sample can comprise, or can be obtained from, any of a variety of cells, tissues, organs, or acellular fluids.
  • Suitable sample sources include eukaryotic cells, bacterial cells, and archaeal cells.
  • Suitable sample sources include single-celled organisms and multi-cellular organisms.
  • Suitable sample sources include singlecell eukaryotic organisms; a plant or a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C.
  • a fungal cell e.g., a yeast cell
  • an animal cell, tissue, or organ e.g. fruit fly, cnidarian, echinoderm, nematode, an insect, an arachnid, etc.
  • a cell, tissue, fluid, or organ from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal); a cell, tissue, fluid, or organ from a mammal (e.g., a human; a non-human primate; an ungulate; a feline; a bovine; an ovine; a caprine; etc.).
  • Suitable sample sources include nematodes, protozoans, and the like.
  • Suitable sample sources include parasites such as helminths, malarial parasites, etc.
  • Suitable sample sources include a cell, tissue, or organism of any of the six kingdoms, e.g., Bacteria (e.g., Eubacteria); Archaebacteria; Protista; Fungi; Plantae; and Animalia.
  • Bacteria e.g., Eubacteria
  • Archaebacteria e.g., Protista
  • Fungi e.g., Plantae
  • Animalia e.g., Animalia.
  • Suitable sample sources include plant-like members of the kingdom Protista, including, but not limited to, algae (e.g., green algae, red algae, glaucophytes, cyanobacteria); fungus-like members of Protista, e.g., slime molds, water molds, etc.; animal-like members of Protista, e.g., flagellates (e.g., Euglena), amoeboids (e.g., amoeba), sporozoans (e.g, Apicomplexa, Myxozoa, Microsporidia), and ciliates (e.g., Paramecium).
  • algae e.g., green algae, red algae, glaucophytes, cyanobacteria
  • fungus-like members of Protista e.g., slime molds, water molds, etc.
  • animal-like members of Protista e.g., flagellates (e.g., Euglen
  • Suitable sample sources include include members of the kingdom Fungi, including, but not limited to, members of any of the phyla: Basidiomycota (club fungi; e.g., members of Agaricus, Amanita, Boletus, Cantherellus, etc.); Ascomycota (sac fungi, including, e.g., Saccharomyces); Mycophycophyta (lichens); Zygomycota (conjugation fungi); and Deuteromycota.
  • Basidiomycota club fungi; e.g., members of Agaricus, Amanita, Boletus, Cantherellus, etc.
  • Ascomycota fungi, including, e.g., Saccharomyces
  • Mycophycophyta lichens
  • Zygomycota conjuggation fungi
  • Deuteromycota Deuteromycota.
  • Suitable sample sources include include members of the kingdom Plantae, including, but not limited to, members of any of the following divisions: Bryophyta (e.g., mosses), Anthocerotophyta (e.g., hornworts), Hepaticophyta (e.g., liverworts), Lycophyta (e.g., club mosses), Sphenophyta (e.g., horsetails), Psilophyta (e.g., whisk ferns), Ophioglossophyta, Pterophyta (e.g., ferns), Cycadophyta, Gingkophyta, Pinophyta, Gnetophyta, and Magnoliophyta (e.g., flowering plants).
  • Bryophyta e.g., mosses
  • Anthocerotophyta e.g., hornworts
  • Hepaticophyta e.g.
  • Suitable sample sources include include members of the kingdom Animalia, including, but not limited to, members of any of the following phyla: Porifera (sponges); Placozoa; Orthonectida (parasites of marine invertebrates); Rhombozoa; Cnidaria (corals, anemones, jellyfish, sea pens, sea pansies, sea wasps); Ctenophora (comb jellies); Platyhelminthes (flatworms); Nemertina (ribbon worms); Ngathostomulida (jawed worms)p Gastrotricha; Rotifera; Priapulida; Kinorhyncha; Loricifera; Acanthocephala; Entoprocta; Nemotoda; Nematomorpha; Cycliophora; Mollusca (mollusks); Sipuncula (peanut worms); Annelida (segmented worms); Tardigrada (water bears); Onychophor
  • Suitable members of Chordata include any member of the following subphyla: Urochordata (sea squirts; including Ascidiacea, Thaliacea, and Larvacea); Cephalochordata (lancelets); Myxini (hagfish); and Vertebrata, where members of Vertebrata include, e.g., members of Petromyzontida (lampreys), Chondrichthyces (cartilaginous fish), Actinopterygii (ray-finned fish), Actinista (coelocanths), Dipnoi (lungfish), Reptilia (reptiles, e.g., snakes, alligators, crocodiles, lizards, etc.), Aves
  • Suitable plants include any monocotyledon and any dicotyledon.
  • Suitable sources of a sample include cells, fluid, tissue, or organ taken from an organism; from a particular cell or group of cells isolated from an organism; etc.
  • suitable sources include xylem, the phloem, the cambium layer, leaves, roots, etc.
  • suitable sources include particular tissues (e.g., lung, liver, heart, kidney, brain, spleen, skin, fetal tissue, etc.), or a particular cell type (e.g., neuronal cells, epithelial cells, endothelial cells, astrocytes, macrophages, glial cells, islet cells, T lymphocytes, B lymphocytes, etc.).
  • tissues e.g., lung, liver, heart, kidney, brain, spleen, skin, fetal tissue, etc.
  • a particular cell type e.g., neuronal cells, epithelial cells, endothelial cells, astrocytes, macrophages, glial cells, islet cells, T lymphocytes, B lymphocytes, etc.
  • the source of the sample is a diseased cell, fluid, tissue, or organ.
  • the source of the sample is a normal (non-diseased) cell, fluid, tissue, or organ.
  • the source of the sample is a pathogen-infected cell, tissue, or organ.
  • Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, Schistosoma parasites, and the like.
  • Helminths include roundworms, heartworms, and phytophagous nematodes (Nematoda), flukes (Tematoda), Acanthocephala, and tapeworms (Cestoda).
  • Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis.
  • pathogens such as parasitic/protozoan pathogens include, but are not limited to: Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi and Toxoplasma gondii.
  • Fungal pathogens include, but are not limited to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.
  • Pathogenic viruses include, e.g., immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like.
  • immunodeficiency virus e.g., HIV
  • influenza virus dengue; West Nile virus; herpes virus; yellow fever virus
  • Hepatitis Virus C Hepatitis Virus A
  • Hepatitis Virus B Hepatitis Virus B
  • papillomavirus papillomavirus
  • Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus, varicella-zoster virus, hepatitis
  • the sample comprises cancer cells.
  • a subject method includes a step of measuring (e.g., measuring a detectable signal produced by CRISPR-Cas effector protein (e.g., Cas13, e.g., Cas13a) -mediated cleavage.
  • CRISPR-Cas effector protein e.g., Cas13, e.g., Cas13a
  • a detectable signal can be any signal that is produced when nucleic acid (e.g., RNA) is cleaved.
  • the step of measuring can include one or more of: gold nanoparticle-based detection (e.g., see Xu et al., Angew Chem Int Ed Engl.
  • a phosphatase to generate a pH change after cleavage reactions, by opening 2’-3’ cyclic phosphates, and by releasing inorganic phosphate into solution), and detection of a labeled detector nucleic acid (e.g., detector RNA or detector DNA) (see below for more details).
  • a labeled detector nucleic acid e.g., detector RNA or detector DNA
  • the readout of such detection methods can be any convenient readout.
  • Examples of possible readouts include but are not limited to: a measured amount of detectable fluorescent signal; a visual analysis of bands on a gel (e.g., bands that represent cleaved product versus uncleaved substrate), a visual or sensor based detection of the presence or absence of a color (i.e., color detection method), and the presence or absence of (or a particular amount of) an electrical signal.
  • the measuring can in some cases be quantitative, e.g., in the sense that the amount of signal detected can be used to determine the amount of actNucleicAcid (e.g., actRNA, actDNA) present in the sample.
  • the measuring can in some cases be qualitative, e.g., in the sense that the presence or absence of detectable signal can indicate the presence or absence of actNucleicAcid.
  • a detectable signal will not be present (e.g., above a given threshold level) unless the actNucleicAcid is present above a particular threshold concentration.
  • the threshold of detection can be titrated by modifying the amount of protein, guide RNA, sample volume, and/or detector nucleic acid (e.g, detector RNA) (if one is used).
  • detector nucleic acid e.g, detector RNA
  • a number of controls can be used if desired in order to set up one or more reactions, each set up to detect a different threshold level of actNucleic Acid (e.g., actRNA, actDNA), and thus such a series of reactions could be used to determine the amount of actNucleic Acid (e.g., actRNA, actDNA) present in a sample (e.g., one could use such a series of reactions to determine that a actNucleic Acid (e.g., actRNA, actDNA) is present in the sample ‘at a concentration of at least X’).
  • actNucleic Acid e.g., actRNA, actDNA
  • a subject method includes also contacting the sample (e.g., a sample comprising a actNucleic Acid (e.g., actRNA, actDNA) and a plurality of non- actNucleic Acid (e.g., actRNA, actDNA)s) with a labeled detector nucleic acid (e.g., detector RNA).
  • a sample e.g., a sample comprising a actNucleic Acid (e.g., actRNA, actDNA) and a plurality of non- actNucleic Acid (e.g., actRNA, actDNA)s
  • a labeled detector nucleic acid e.g., detector RNA
  • a subject method includes contacting a sample with a labeled detector nucleic acid (e.g., detector RNA) comprising a fluorescence-emitting dye pair; the CRISPR-Cas effector protein cleaves the labeled detector nucleic acid (e.g., detector RNA) after it is activated; and the detectable signal that is measured is produced by the fluorescence-emitting dye pair.
  • a subject method includes contacting a sample with a labeled detector nucleic acid (e.g., detector RNA) comprising a fluorescence resonance energy transfer (FRET) pair or a quencher/fluor pair, or both.
  • FRET fluorescence resonance energy transfer
  • a subject method includes contacting a sample with a labeled detector nucleic acid (e.g., detector RNA) comprising a FRET pair. In some cases, a subject method includes contacting a sample with a labeled detector nucleic acid (e.g., detector RNA) comprising a fluor/quencher pair. Fluorescence-emitting dye pairs comprise a FRET pair or a quencher/fluor pair. In both cases of a FRET pair and a quencher/fluor pair, the emission spectrum of one of the dyes overlaps a region of the absorption spectrum of the other dye in the pair.
  • fluorescence-emitting dye pair is a generic term used to encompass both a “fluorescence resonance energy transfer (FRET) pair” and a “quencher/fluor pair,” both of which terms are discussed in more detail below.
  • FRET fluorescence resonance energy transfer
  • quencher/fluor pair both of which terms are discussed in more detail below.
  • fluorescenceemitting dye pair is used interchangeably with the phrase “a FRET pair and/or a quencher/fluor pair.”
  • the labeled detector nucleic acid e.g., detector RNA
  • the labeled detector nucleic acid produces an amount of detectable signal prior to being cleaved, and the amount of detectable signal that is measured is reduced when the labeled detector nucleic acid (e.g., detector RNA) is cleaved.
  • the labeled detector nucleic acid (e.g., detector RNA) produces a first detectable signal prior to being cleaved (e.g., from a FRET pair) and a second detectable signal when the labeled detector nucleic acid (e.g., detector RNA) is cleaved (e.g., from a quencher/fluor pair).
  • the labeled detector nucleic acid (e.g., detector RNA) comprises a FRET pair and a quencher/fluor pair.
  • the labeled detector nucleic acid (e.g., detector RNA) comprises a FRET pair.
  • FRET is a process by which radiationless transfer of energy occurs from an excited state fluorophore to a second chromophore in close proximity. The range over which the energy transfer can take place is limited to approximately 10 nanometers (100 angstroms), and the efficiency of transfer is extremely sensitive to the separation distance between fluorophores.
  • FRET fluorescence resonance energy transfer
  • FRET fluorescence resonance energy transfer
  • the donor-acceptor pair (a FRET donor moiety and a FRET acceptor moiety) is referred to herein as a “FRET pair” or a “signal FRET pair.”
  • a subject labeled detector nucleic acid e.g., detector RNA
  • detector nucleic acid includes two signal partners (a signal pair), when one signal partner is a FRET donor moiety and the other signal partner is a FRET acceptor moiety.
  • a subject labeled detector nucleic acid e.g., detector RNA
  • a FRET pair a FRET donor moiety and a FRET acceptor moiety
  • a detectable signal a FRET signal
  • the signal partners are in close proximity (e.g., while on the same RNA molecule), but the signal will be reduced (or absent) when the partners are separated (e.g., after cleavage of the RNA molecule by a Cas13 protein).
  • FRET donor and acceptor moieties will be known to one of ordinary skill in the art and any convenient FRET pair (e.g., any convenient donor and acceptor moiety pair) can be used. Examples of suitable FRET pairs include but are not limited to those presented in Table 2. See also: Bajar et al. Sensors (Basel). 2016 Sep 14;16(9); and Abraham et al. PLoS One. 2015 Aug 3;10(8):e0134436.
  • a detectable signal is produced when the labeled detector nucleic acid (e.g., detector RNA) is cleaved (e.g., in some cases, the labeled detector nucleic acid (e.g., detector RNA) comprises a quencher/fluor pair.
  • the labeled detector nucleic acid e.g., detector RNA
  • the labeled detector nucleic acid comprises a quencher/fluor pair.
  • One signal partner of a signal quenching pair produces a detectable signal and the other signal partner is a quencher moiety that quenches the detectable signal of the first signal partner (i.e., the quencher moiety quenches the signal of the signal moiety such that the signal from the signal moiety is reduced (quenched) when the signal partners are in proximity to one another, e.g., when the signal partners of the signal pair are in close proximity).
  • an amount of detectable signal increases when the labeled detector nucleic acid (e.g., detector RNA) is cleaved.
  • the signal exhibited by one signal partner a signal moiety
  • the other signal partner a quencher signal moiety
  • one signal partner e.g., the first signal partner
  • quenching pair is a signal moiety that produces a detectable signal that is quenched by the second signal partner (e.g., a quencher moiety).
  • the signal partners of such a quencher/fluor pair will thus produce a detectable signal when the partners are separated (e.g., after cleavage of the detector nucleic acid (e.g., detector RNA) by a CRISPR-Cas effector protein), but the signal will be quenched when the partners are in close proximity (e.g., prior to cleavage of the detector nucleic acid (e.g., detector RNA) by a CRISPR-Cas effector protein).
  • a quencher moiety can quench a signal from the signal moiety (e.g., prior to cleave of the detector nucleic acid (e.g., detector RNA) by a CRISPR-Cas effector protein) to various degrees.
  • a quencher moiety quenches the signal from the signal moiety where the signal detected in the presence of the quencher moiety (when the signal partners are in proximity to one another) is 95% or less of the signal detected in the absence of the quencher moiety (when the signal partners are separated).
  • the signal detected in the presence of the quencher moiety can be 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the signal detected in the absence of the quencher moiety. In some cases, no signal (e.g., above background) is detected in the presence of the quencher moiety.
  • the signal detected in the absence of the quencher moiety (when the signal partners are separated) is at least 1 .2 fold greater (e.g., at least 1 .3f old , at least 1 .5 fold, at least 1 .7 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 20 fold, or at least 50 fold greater) than the signal detected in the presence of the quencher moiety (when the signal partners are in proximity to one another).
  • the signal detected in the absence of the quencher moiety is at least 1 .2 fold greater (e.g., at least 1 .3f old , at least 1 .5 fold, at least 1 .7 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 20 fold, or at least 50 fold greater) than the signal detected
  • the signal moiety is a fluorescent label.
  • the quencher moiety quenches the signal (the light signal) from the fluorescent label (e.g., by absorbing energy in the emission spectra of the label).
  • the emission (the signal) from the fluorescent label is detectable because the signal is not absorbed by the quencher moiety.
  • Any convenient donor acceptor pair (signal moiety /quencher moiety pair) can be used and many suitable pairs are known in the art.
  • the quencher moiety absorbs energy from the signal moiety (also referred to herein as a “detectable label”) and then emits a signal (e.g., light at a different wavelength).
  • the quencher moiety is itself a signal moiety (e.g., a signal moiety can be 6-carboxyfluorescein while the quencher moiety can be 6-carboxy-tetramethylrhodamine), and in some such cases, the pair could also be a FRET pair.
  • a quencher moiety is a dark quencher. A dark quencher can absorb excitation energy and dissipate the energy in a different way (e.g., as heat).
  • a dark quencher has minimal to no fluorescence of its own (does not emit fluorescence). Examples of dark quenchers are further described in U.S. patent numbers 8,822,673 and 8,586,718; U.S. patent publications 20140378330, 20140349295, and 20140194611 ; and international patent applications: WO200142505 and WO200186001 , all if which are hereby incorporated by reference in their entirety.
  • fluorescent labels include, but are not limited to: an Alexa Fluor® dye, an ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11 , ATTO Rho12, ATTO Thiol 2, ATTO Rho101 , ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740), a DyLight dye, a cyanine dye (e.g., Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), a Fluor 390, AT
  • a detectable label is a fluorescent label selected from: an Alexa Fluor® dye, an ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11 , ATTO Rho12, ATTO Thiol 2, ATTO Rho101 , ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740), a DyLight dye, a cyanine dye (e.g., Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy
  • a detectable label is a fluorescent label selected from: an Alexa Fluor® dye, an ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11 , ATTO Rho12, ATTO Thiol 2, ATTO Rho101 , ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740), a DyLight dye, a cyanine dye (e.g., Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy
  • ATTO dyes include, but are not limited to: ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11 , ATTO Rho12, ATTO Thiol 2, ATTO Rho101 , ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, and ATTO 740.
  • AlexaFluor dyes include, but are not limited to: Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 635, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, Alexa Fluor® 790, and the like.
  • quencher moieties include, but are not limited to: a dark quencher, a Black Hole Quencher® (BHQ®) (e.g., BHQ-0, BHQ-1 , BHQ-2, BHQ-3), a Qxl quencher, an ATTO quencher (e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q), dimethylaminoazobenzenesulfonic acid (Dabsyl), Iowa Black RQ, Iowa Black FQ, IRDye QC-1 , a QSY dye (e.g., QSY 7, QSY 9, QSY 21 ), AbsoluteQuencher, Eclipse, and metal clusters such as gold nanoparticles, and the like.
  • BHQ® Black Hole Quencher®
  • BHQ® Black Hole Quencher®
  • ATTO quencher e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q
  • Dabsyl dimethylamino
  • a quencher moiety is selected from: a dark quencher, a Black Hole Quencher® (BHQ®) (e.g., BHQ-0, BHQ-1 , BHQ-2, BHQ-3), a Qxl quencher, an ATTO quencher (e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q), dimethylaminoazobenzenesulfonic acid (Dabsyl), Iowa Black RQ, Iowa Black FQ, IRDye QC-1 , a QSY dye (e.g., QSY 7, QSY 9, QSY 21 ), AbsoluteQuencher, Eclipse, and a metal cluster.
  • BHQ® Black Hole Quencher®
  • BHQ® Black Hole Quencher®
  • ATTO quencher e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q
  • Dabsyl dimethylaminoazobenzenesulfonic acid
  • Examples of an ATTO quencher include, but are not limited to: ATTO 540Q, ATTO 580Q, and ATTO 612Q.
  • Examples of a Black Hole Quencher® (BHQ®) include, but are not limited to: BHQ-0 (493 nm), BHQ-1 (534 nm), BHQ-2 (579 nm) and BHQ-3 (672 nm).
  • detectable labels e.g., fluorescent dyes
  • quencher moieties see, e.g., Bao et al., Annu Rev Biomed Eng. 2009;11 :25-47; as well as U.S. patent numbers 8,822,673 and 8,586,718; U.S. patent publications 20140378330, 20140349295, 20140194611 , 20130323851 , 20130224871 , 20110223677, 20110190486, 20110172420, 20060179585 and 20030003486; and international patent applications: WQ200142505 and WO200186001 , all of which are hereby incorporated by reference in their entirety.
  • cleavage of a labeled detector nucleic acid can be detected by measuring a colorimetric read-out.
  • the liberation of a fluorophore e.g., liberation from a FRET pair, liberation from a quencher/fluor pair, and the like
  • cleavage of a subject labeled detector nucleic acid can be detected by a color-shift.
  • Such a shift can be expressed as a loss of an amount of signal of one color (wavelength), a gain in the amount of another color, a change in the ration of one color to another, and the like.
  • detector nucleic acids and the use of CRISPR systems with trans cleavage activity, e.g., for nucleic acid detection, see, e.g., Huang et al., Biosensors (Basel). 2022 Sep 20;12(10):779; and Feng et al., Anal Chem. 2023 Jan 10;95(1):206-217.
  • kits/systems for carrying out a subject method comprise various combinations of components useful in any of the methods described elsewhere herein.
  • a kit can further include one or more additional reagents, where such additional reagents can be any convenient reagent.
  • Components of a subject kit can be in separate containers; or can be combined in a single container. In some cases one or more of a kit’s components are pharmaceutically formulated for administration to a human.
  • a subject kit can further include instructions for using the components of the kit to practice the subject methods (e.g., dosing instructions, instructions to administer the component(s) to an individual.
  • the instructions for practicing the subject methods are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e. , associated with the packaging or subpackaging) etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
  • a method of activating a CRISPR-Cas effector protein with a nucleic acid of interest comprising: contacting a capture nucleic acid (capNucleicAcid) with a CRISPR- Cas effector protein and a CRISPR-Cas guide RNA in the presence of a nucleic acid of interest (actNucleicAcid), wherein:
  • the capNucleicAcid comprises: an anchor region that hybridizes to the CRISPR-Cas guide RNA, and a capture region that hybridizes to the actNucleicAcid;
  • the CRISPR-Cas guide RNA comprises: a protein-binding region that binds to the CRISPR-Cas effector protein, and an 8-15 nucleotide (nt) guide sequence that hybridizes with the anchor region of the capNucleicAcid; and
  • a method of detecting a nucleic acid of interest in a sample comprising:
  • a CRISPR-Cas guide RNA comprising: a protein-binding region that binds to the CRISPR-Cas effector protein, and an 8-15 nucleotide (nt) guide sequence that hybridizes with an anchor region of a capture nucleic acid (capNucleicAcid);
  • the capNucleicAcid which comprises: said anchor region that hybridizes to the CRISPR-Cas guide RNA, and a capture region that hybridizes to a nucleic acid of interest (actNucleicAcid);
  • actRNA RNA
  • actNucleicAcid is a DNA (actDNA).
  • capNucleicAcid is an RNA (capRNA).
  • capNucleicAcid is a DNA (capDNA).
  • capRNA comprises a 5’ tail region that is positioned 5’ of the capture region.
  • capRNA comprises a 3’ region that is positioned 3’ of the anchor region.
  • the 3’ region is 1-10 nt long.
  • detecting comprises: gold nanoparticle-based detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, fluorescent signal detection, semiconductor-based sensing, or any combination thereof.
  • the labeled single stranded detector nucleic acid comprises one or more non-natural internucleoside linkages, one or more nucleic acid mimetics, one or more modified sugar moieties, one or more modified nucleobases, one or more locked nucleic acids (LNAs), one or more peptide nucleic acids (PNAs), one or more morpholino nucleic acids, one or more cyclohexenyl nucleic acids (CeNAs), or any combination thereof.
  • the actNucleicAcid is from a virus selected from: Zika virus, human immunodeficiency virus (HIV), hepatitis B virus, hepatitis C virus, herpes virus, herpes simplex virus I, herpes simplex virus II, papillomavirus, rabies virus, cytomegalovirus, human serum parvo- like virus, respiratory syncytial virus, varicella-zoster virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, west Nile virus,
  • actNucleicAcid is from pathogenic bacteria selected from: Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, and Brucella abortus.
  • pathogenic bacteria selected from: Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Ne
  • kits for detecting a nucleic acid of interest comprising:
  • a CRISPR-Cas guide RNA comprising: a protein-binding region that can bind to a CRISPR-Cas effector protein, and an 8-15 nucleotide (nt) guide sequence that can hybridize with an anchor region of a capture nucleic acid (capNucleicAcid);
  • capNucleicAcid which comprises: said anchor region that can hybridize to the CRISPR-Cas guide RNA, and a capture region that can hybridize to a nucleic acid of interest (actNucleicAcid).
  • kit of 43 further comprising: a labeled single stranded detector nucleic acid that does not hybridize with the CRISPR-Cas guide RNA.
  • kits of 43 or 44 further comprising the CRISPR-Cas effector protein or a nucleic acid encoding the CRISPR-Cas effector protein.
  • 46. The kit of any one of 43-45, further comprising a positive control actNucleicAcid.
  • kits of any one of 43-50, wherein the CRISPR-Cas effector protein is a Cas13 protein or a Cas 12 protein.
  • Example 1 miRNA detection with CRISPR-Cas13a using a split guide RNA Results
  • FIG.1 Cas13a activated by ssRNA ⁇ 20nt with a split gRNA.
  • A Schematic illustrating ssRNA detection by Cas13a. Cas13a (light blue) complexes with a gRNA (hot pink) to form a Cas13a ribonucleoprotein complex (RNP). The RNP binds to target RNA (grey), activates, and cleaves quenched fluorophore reporters.
  • B Top: Schematic illustrating a Cas13a full-length gRNA with a 20nt seed region (hot pink) complemented with a 10nt (yellow), 20nt (green), and 36nt (grey) target RNA.
  • the split gRNA complements to the full-length target RNA (36nt, with 20nt sequence complementary to the seed region, shown in grey).
  • Target RNA is at 1e6 copies/uL in the final reaction.
  • Both parts of the split gRNA are at 25nM in the reaction, equimolar to the Cas13a.
  • Full-length gRNA is also at 25nM. Mean with three technical replicates is plotted.
  • FIG.6 Supporting data for Figure 1.
  • A Fluorescence intensity time course data for full-length gRNA and 36nt target RNA at two-fold dilutions ranging from 5e6 copies/uL to 1 .6e5 copies/uL in the final reaction. Each concentration of target RNA has three technical replicates; each replicate is plotted separately. Exponential or linear regression fit to data. Slope of the linear region is calculated and plotted in FIG. 1 B. Guide RNA equimolar to Cas13a at 25nM.
  • B Scatter plot showing reaction rate (AU/s) for Cas13a RNPs activated by target RNA of different lengths and concentrations.
  • Activation of Cas13a RNP is tested with 10nt target A or B at 1e8 copies/uL or both at 5e7 copies/uL each, for a total of 1e8 copies/uL of total RNA (A, yellow, complementary to the first 10nt of the gRNA seed region; B, pink, complementary to the final 10nt of the seed region).
  • Activity with 10nt targets is compared to known activators: 20nt target RNA (green bar, squares, 1 e7 copies/uL) and 36nt target RNA (grey bar, circles, 1e6 copies/uL).
  • Reaction rate is calculated as the slope of the linear region of the reaction progress curve (AU vs. time).
  • Cas13a and gRNA are equimolar at 25nM.
  • Stats For B we used pairwise analysis of the reaction rates between experimental conditions and negative controls using a Brown- Forsythe ANOVA to account for variation in standard deviation and Dunnet’s T3 test for multiple comparisons. We report multiplicity- adjusted p-values. Non-significant interactions (p>0.05), * is p ⁇ 0.05, “ is p ⁇ 0.01.
  • split gRNA system For the split gRNA system to be a useful strategy to detect short ssRNA, it must work when the short ssRNA that completes the split gRNA is the limiting reagent.
  • FIG. 1 D we showed that the split guide strategy works when both parts of the split gRNA were equimolar to the Cas13a and the target RNA was the limiting reagent.
  • the split guide strategy when the short ssRNA was limiting. To clarify the new roles of the RNAs in the Cas13a split guide system (FIG.
  • the short ssRNA of interest the activating RNA, or ‘actRNA’ (yellow) and the remaining portion of the standard gRNA the ‘split gRNA’ (hot pink).
  • the split gRNA contains the 30nt stem-loop region and a portion of the seed region that we call the ‘anchor region’ (light pink).
  • the target RNA from the conventional guide system is now called the capture RNA, or ‘capRNA' (grey). It is added to the split guide system and fully complements to the anchor region of the split gRNA, becoming a part of the RNP.
  • the capRNA also contains a sequence complementary to the actRNA (short ssRNA of interest), which we call the ‘capture region’ (yellow). The capRNA thus holds the split gRNA and actRNA together, completing the dsRNA seed region.
  • capRNA 1 shown in blue with two asterisks
  • capRNA 2 a new 36nt capRNA
  • CapRNAs 1 and 2 had drastically different levels of background activity (1 .2 ⁇ SEM 0.1 AU/s and 0.08 ⁇ SEM 0.01 AU/s, respectively) but similar levels of activity in the presence of the 10nt actRNA (1 .5 ⁇ SD 0.04 AU/s and 1 .6 ⁇ SEM 0.1 AU/s, respectively), such that their background-subtracted reaction rates were significantly different (adjusted p- value of 0.0050; FIG. 7C).
  • capRNA 2 had such a high background that the positive reaction rate was not significantly different from the background (adjusted p-value of 0.0503).
  • capRNA 4 (7nt 5’ tail) performed significantly better than sequence-matched capRNA 2 (11 nt 5’ tail) and capRNA 3 (18nt 5’ tail) but performed similarly to capRNA 1 (original 11 nt 5’ tail) (FIG. 7C).
  • capRNA 1 was tested to evaluate the importance of the 3’ flanking sequence and the 5’ tail and found that deleting either 3’ flank or 5’ tail reduced the 10nt actRNA reaction rate (FIG. 7D).
  • capRNA 1 With capRNA 1 at 2.5nM, our estimated limit of detection for a 10nt actRNA is 1 .2e5 copies/uL (FIG. 2E), significantly better than our inability to detect a 10nt target RNA with a full-length gRNA (FIG. 1 B). Unless otherwise noted, we use capRNA 1 for subsequent experiments.
  • FIG. 2 Cas13a detects 10nt ssRNA with split guide system.
  • A Schematic illustrating the standard full-length guide system (left) and introducing the split guide system and nomenclature (right). The full-length gRNA is split in two in the seed region. The first part of the gRNA is referred to as the split gRNA (hot pink). The second part of the gRNA is now called the activating RNA, or actRNA (yellow); it is the limiting reagent of the system.
  • the target RNA becomes the capture RNA, or capRNA. The capRNA complements to the split gRNA via the anchor region (light pink) and the actRNA via the capture region (pale yellow).
  • capRNA 1 concentration was tested ranging from 25nM to 17pM. At each capRNA concentration, the reaction was tested with actRNA (yellow bars, triangles) or without actRNA (grey bars, empty circles). Cas13a and split gRNA are at 25nM. 10nt actRNA is at 1 e6 copies/uL in the final reaction. For the full-length guide system, gRNA is equimolar to Cas13a at 25nM and 36nt target is at 1 e6 copies/uL. Mean reaction rate (AU/s) with three technical replicates plotted.
  • D Top: Schematic illustrating the split guide system with split gRNA (1 Ont anchor) and 10nt actRNA bound to different capRNAs.
  • the sequence of the anchor and capture regions are held constant, but the length of the 5’ tail of capRNA varies.
  • capRNAs 3, 2, and 4 shown in light grey with 18nt, 11 nt, and 7nt 5’ tails, respectively, the sequences of the 5’ tail and 3’ flanking sequence are constant.
  • capRNA 1 shown in blue with an 11 nt 5’ tail, the sequences of the 5’ tail and 3’ flanking sequences are different from the length-matched capRNA 2.
  • gRNA is equimolar to Cas13a at 25nM and 36nt target is at 1 e6 copies/uL.
  • E Limit of detection of 10nt actRNA determined with optimized reaction conditions. LOD of 1 Ont actRNA was tested with Cas13a equimolar to split gRNA (1 Ont anchor) at 25nM and capRNA 1 at 2.5nM. Concentration of 10nt actRNA tested at 2-fold dilutions from 1e7 copies/uL to 4e4 copies/uL.
  • FIG. 7 Supporting Data for Figure 2.
  • Reaction rate (AU/s) for split guide or full-length guide system with the mean background activity subtracted (Cas13a RNP and capRNA alone for split guide system, or Cas13a RNP alone for the full-length guide system).
  • Cas13a, split gRNA, and capRNA 1 are equimolar at 25nM.
  • 10nt actRNA at 1 e6 copies/uL.
  • Cas13a and split gRNA are equimolar at 25nM. Concentration of capRNA 1 varies from 25nM to 17pM. 10nt actRNA at 1 e6 copies/uL.
  • gRNA is equimolar to Cas13a at 25nM and 36nt target is at 1 e6 copies/uL.
  • C Supporting figure for Fig. 2d.
  • D Left: Schematic illustrating the split guide system with split gRNA (1 Ont anchor) and 10nt actRNA bound to different capRNAs.
  • FIG. 8 Predicted structures for split gRNA-capRNA complex.
  • Predicted complex structure with same split gRNA (10nt anchor region) and varying capRNAs.
  • Split gRNA highlighted in pink.
  • anchor region highlighted in pink and capture region is highlighted in yellow.
  • sequence of the 3’ flanking region is such that there is an additional 1 nt of complementary base pairing with the split gRNA, making the anchor region effectively 1 1 nt.
  • Complex structure predicted with the NUPACK web application.
  • split gRNA performance was not dependent on the specific sequences we initially chose and would work with different capture and anchor region sequences.
  • Split gRNA 2 blue
  • the capture regions for the capRNAs in both systems were the same length but different sequences.
  • Split guide system 2 detected both actRNAs above background and showed the same trend as the split system 1 where the 10nt actRNA resulted in higher activity than the 21 nt actRNA (FIG. 3C, bottom, and FIG. 10C). However, when comparing the activity of the 10nt and 21 nt actRNAs relative to the full-length control, the split guide system 2 performed proportionally better than the split guide system 1 .
  • the 10nt actRNA was 46% of the control and the 21 nt actRNA was 22% of the control for split guide system 2, whereas the 10nt actRNA was 27% of the control and the 21 nt actRNA was 11% of the control for split guide system 1 (FIG. 10D).
  • the split guide system can detect actRNA ranging from 8nt to at least 24nt.
  • FIG. 3 Split guide system works across a range of RNA lengths and sequences.
  • A Top: Schematic illustrating the split gRNA (hot pink) with varied length of its anchor region (ranging from 5 to 12nt) and the corresponding actRNA (yellow, ranging from 15 to 8nt) such that the anchor plus capture region remains constant at 20nt.
  • Bottom Reaction rate (AU/s) of different split gRNA and actRNA pairs with the mean background activity of the Cas13a RNP and capRNA alone subtracted.
  • Cas13a, split gRNA, and capRNA 1 are equimolar at 25nM. All actRNA is at 1e7 copies/uL in the final reaction.
  • actRNA may either complement perfectly with a capRNA with a blunt end (1 Ont actRNA with capRNA 5, 17nt actRNA with capRNA 6) or there may be an overhang of the actRNA (17nt actRNA with capRNA 5 has a 7nt overhang of the actRNA) or the capRNA (20nt actRNA with capRNA 7 has a 1 nt overhang of the capRNA).
  • Reaction rate (AU/s) of different actRNA and capRNA pairs with mean background activity of the Cas13a RNP and capRNA alone subtracted. For each actRNA, background- subtracted reaction rate for two different capRNAs is plotted.
  • Split gRNA 1 (hot pink) with actRNA 1 (yellow) and capRNA 1 (grey) are the same sequence as previously tested in this paper.
  • Split gRNA 2 (blue) with actRNA 2 (blue) and capRNA 8 (grey) are distinct RNA sequences from split guide system 1. Both systems are tested with a 10nt and 21 nt actRNA and compared to their sequence-matched full-length gRNA variants.
  • Split gRNA 1 is tested with a 10nt and 21 nt actRNA and compared to full-length gRNA 1 with a 36nt target. The same is shown for split gRNA 2 and full-length gRNA 2. All reaction rates are reported with mean background activity subtracted.
  • Cas13a is equimolar to split gRNA at 25nM, capRNA is at 2.5nM, and actRNA is at 1 e6 copies/uL.
  • Cas13a is equimolar to gRNA at 25nM, and target RNA is at 1 e6 copies/uL.
  • Stats In B for each actRNA, the background-subtracted reaction rate is compared between the two capRNA conditions using a Brown- Forsythe ANOVA to account for variation in standard deviation and Dunnet's T3 test for multiple comparisons.
  • FIG. 9 Supporting Data for Figure 3.
  • A Supporting figure for FIG. 3A. For each split gRNA species, the reaction was tested with corresponding actRNA (yellow bars, rhombuses) or without actRNA (grey bars, empty circles). Cas13a, split gRNAs, and capRNA 1 are equimolar at 25nM. All actRNA is at 1e7 copies/uL in the final reaction. Mean reaction rate (AU/s) with three technical replicates plotted.
  • B The melting temperature (°C) of the capture region versus anchor region is plotted for each split gRNA and actRNA pair. For a given pair, if the signal was detected over background (see A), the dot is colored pink.
  • FIG. 10 Comparison of two sequence-unique split guide systems.
  • A,B Predicted complex structure for gRNA system 1 (A) and 2 (B). Top row is full- length gRNA bound to a 36nt target. Middle row is split gRNA bound to capRNA. Bottom row is split gRNA and 21 nt actRNA bound to capRNA. Stability of complex reported as free energy (AG, kcal/mol). Complex structures and free energy predicted with the NUPACK web application.
  • C Supporting figure for FIG. 3C. Reaction rates (AU/s) for gRNA system 1 and 2, either split gRNA paired with 21 nt or 10nt actRNA or full-length gRNA paired with a 36nt target.
  • Cas13a is equimolar to split gRNA at 25nM, capRNA is at 2.5nM, and actRNA is at 1 e6 copies/uL.
  • Cas13a is equimolar to gRNA at 25nM, and target RNA is at 1 e6 copies/uL.
  • split guide system is specific against RNA mismatches and misalignments [00237]
  • the Cas13a split guide system To be a useful strategy for molecular diagnostics and RNA discovery, it must demonstrate target specificity. We sought to characterize the specificity of the system to on-target vs off-target actRNA, looking at mismatches between the actRNA and capRNA, and extensions and truncations of the actRNA.
  • FIG. 4 Split guide system is specific against RNA mismatches and misalignments.
  • A Top: Schematic illustrating test of mismatch sensitivity. The split gRNA (hot pink, 10nt anchor) and 17nt actRNA (yellow) containing a 2nt mismatch tiled across the first 10nt of the actRNA are bound to capRNA 5. The capRNA contains a 10nt anchor region and a 10nt capture region, such that only the first 10nt of the actRNA bind to the capRNA, leaving 7nt unbound on the 3’ end of the actRNA.
  • Bottom Split guide system is tested with 17nt actRNA containing 2nt mismatches.
  • Cas13a is equimolar to split gRNA at 25nM, capRNA 5 is at 2.5nM, and all actRNA is at 1 e7 copies/uL. Mean reaction rate (AU/s) is plotted with three technical replicates. All reactions are compared to the actRNA with no mismatches.
  • B Top: Schematic illustrating test of overlap sensitivity. Split gRNA (hot pink, 10nt anchor) is paired with actRNA (yellow) ranging from 10 to 15nt, with the actRNA overlapping the anchor region of the split gRNA up to 5nt. The region of the actRNA that overlaps the split gRNA has the same sequence as the split gRNA. The split gRNA and actRNA complement to capRNA 1 .
  • split gRNA hot pink, 10nt anchor
  • actRNA yellow
  • actRNA yellow
  • a 10nt actRNA with no gap and an 8nt actRNA with a 2nt gap between the 3’ end of the split gRNA and the 5’ end of the actRNA. All are in complex with capRNA 1 .
  • the bottom schematic shows the same split gRNA adjacent to a 17nt actRNA. Multiple 17nt actRNAs are tested with a gap between the 3’ end of the split gRNA and the 5’ end of the actRNA. The gap size ranges from Ont to 4nt.
  • the split gRNA and 17nt actRNAs are in complex with capRNA 6 containing a 17nt capture region.
  • FIG. 11 Supporting Data for Figures 4 and 5.
  • Top Schematic illustrating further testing of mismatch sensitivity.
  • the split gRNA (hot pink, 10nt anchor) and 17nt actRNA (yellow) containing a 2nt mismatch tiled across the first 10nt of the actRNA are bound to capRNA 6.
  • CapRNA 6 contains a 10nt anchor region and a 17nt capture region, such that the entire actRNA binds to the capRNA.
  • Bottom Reaction rates (AU/s) for all mismatch actRNAs paired with capRNA 6.
  • Cas13a is equimolar to split gRNA at 25nM, capRNA 6 is at 2.5nM, and all actRNA is at 1e7 copies/uL. Mean reaction rate (AU/s) is plotted with three technical replicates. All reactions are compared to the actRNA with no mismatches.
  • B Test of synthetic 17nt actRNA detection in total cell RNA background. Cell RNA, extracted from Lenti-X HEK 293T cells (grey bars), Jurkat (pink bars), or HL- 60 cells (yellow bars), is added at three different concentrations in the final reaction.
  • Cas13a is equimolar to split gRNA at 25nM, capRNA 6 (17nt capture region) is at 2.5nM, and 17nt actRNA is at 1e7 copies/uL.
  • Stats For A we performed a Brown-Forsythe ANOVA to account for variation in standard deviation and Dunnet’s T3 test for follow-up testing of experimental conditions versus the actRNA with no mismatches.
  • capRNA For each capRNA, we added either synthetic miRNA at 1 e7 copies/uL or cell RNA extract from Lenti-X HEK 293T cells at 1 ng/uL (FIG. 5D). For all three miRNA targets, the synthetic miRNAs were detected above background (yellow bars). miR_3178 (17nt) and miR_4505 (18nt) were also detected above background in the cell RNA sample (blue bars). Pre-annealing the capRNA with either the synthetic miRNA or cell RNA extract further boosted the signal for all conditions tested, except miR_31 in total cell RNA extract (FIG. 5E, striped vs solid bars).
  • FIG. 5 Split guide system detects endogenous cellular miRNA.
  • A Schematic illustrating the detection of synthetic actRNA in the presence of total cell RNA. The total cell RNA acts as a general RNA background to which the actRNA of interest is spiked in. Any Cas13a RNP activated by the actRNA will cleave both the fluorescent reporter and the cell RNA.
  • B Limit of detection analysis of 20nt actRNA in a cell RNA background. Cell RNA, extracted from Lenti-X HEK 293T cells, is at 1 ng/uL in the final reaction.
  • C For miRNA detection, screening capRNA for low background signal is critical. Top: Schematic illustrating addition of capRNA to Cas13a RNPs and resultant fluorescent signal generated from RNPs activated by capRNA in the absence of the activating miRNA.
  • CapRNAs tested contain the same 3’ flanking sequence (5nt) and 10nt anchor region. The capture region is unique to each capRNA and perfectly complementary to the miRNA of interest, with 5’ tail.
  • Bottom CapRNA for six miRNA targets tested for background activity. Mean reaction rate (AU/s) of Cas13a RNPs plus capRNA plotted with three technical replicates. Cas13a is equimolar to split gRNA (1 Ont anchor) at 25nM. All capRNA present at 2.5nM. The three capRNAs with the lowest activity were selected for further testing.
  • D Top: Schematic illustrating miRNA detection by split guide system. CapRNA and synthetic miRNA were added to Cas13a RNPs containing a split gRNA (1 Ont anchor) (top schematic).
  • total cell RNA was added in lieu of synthetic miRNA (bottom schematic).
  • mean reaction rate (AU/s) is plotted with three technical replicates for the condition with synthetic miRNA (yellow bars, circles), total cell RNA (blue bars, triangles), and no activating RNA (Cas13a RNP and capRNA only; grey bars, rhombuses).
  • Cas13a is equimolar to split gRNA at 25nM and the capRNA is at 2.5nM.
  • Synthetic miRNA is added at a final concentration of 1e7 copies/uL.
  • Total cell RNA from Lenti-X HEK 293T cells is added at a final concentration of 1 ng/uL. All reactions compared to their specific no target control.
  • E Top: Schematic illustrating use of annealing miRNA and capRNA to improve detection. Synthetic miRNA (box with yellow outline, top) or total cell RNA (box with blue outline, bottom) is annealed with capRNA before adding it to the Cas13a RNP mixture.
  • Bottom Detection of miR_31 and miR_4505 was tested with a pre-annealing step. Mean background- subtracted reaction rate (AU/s) of annealed and unannealed conditions is plotted with three technical replicates.
  • the graph shows the background-subtracted reaction rate of synthetic miRNA (yellow bars), synthetic miRNA annealed to capRNA (striped yellow bars), total cell RNA (blue bars), and total cell RNA annealed to capRNA (striped blue bars).
  • Cas13a is equimolar to split gRNA at 25nM and the capRNA is at 2.5nM.
  • Synthetic miRNA is added at a final concentration of 1 e7 copies/uL.
  • Total cell RNA from Lenti-X HEK 293T cells is added at a final concentration of 1 ng/uL. Detection of miRNA is compared with and without annealing for each miRNA target and source.
  • mean reaction rate (AU/s) is plotted with three technical replicates for the condition with no target (Cas13a RNP and capRNA only; grey bars, rhombuses), synthetic miRNA (yellow bars, circles), and whole cell lysate (pink bars, triangles).
  • Cas13a is equimolar to split gRNA at 25nM and the capRNA is at 2.5nM.
  • Synthetic miRNA is added at a final concentration of 1 e7 copies/uL.
  • Cas13a is equimolar to split gRNA at 25nM and the capRNA is at 2.5nM.
  • Synthetic miRNA is added at a final concentration of 1e7 copies/uL.
  • Whole cell lysate from Lenti-X HEK 293T cells is added at a final concentration of 10 cells/uL.
  • Detection of miRNA is compared with and without annealing for each miRNA target in whole cell lysate.
  • Stats We used pairwise analysis of the reaction rates for each experimental conditions with and without actRNA (D, F middle) or with and without annealing (E, F right) using a Brown-Forsythe ANOVA to account for variation in standard deviation and Dunnet’s T3 test for multiple comparisons.
  • multiplicity-adjusted p-values * is p ⁇ 0.05, ** is p ⁇ 0.01 , *** is p ⁇ 0.001 . See FIG. 11 and FIG. 13 for more details.
  • FIG. 12 Length distribution of validated human miRNAs. Histogram of lengths of all human-annotated miRNAs in miRbase release 22.1.
  • FIG. 13 Predicted structures for split gRNA-capRNA complex for miRNA targets. Supporting data for FIG. 5A. Predicted complex structure for split gRNA bound to capRNA targeting six different cellular miRNAs. The split gRNA is highlighted in pink, with the 10nt anchor region highlighted with a grey box. The capture region (length varies) is highlighted with a yellow box. Stability of complex reported as free energy (AG, kcal/mol). Complex structures and free energy predicted with the NUPACK web application.
  • the Cas13a split guide system uses a gRNA with a short seed region (split gRNA) paired with a capture RNA (capRNA) to detect RNA targets ranging from 8nt to 24nt.
  • split gRNA short seed region
  • capture RNA capture RNA
  • the split guide system is sensitive, even with small targets.
  • the split guide system can detect a 10nt target with an estimated LOD of 1 .2e5 copies/uL (200fM), which is within the range of concentrations that clinically- relevant miRNAs are found in serum (8.9e3 to 1 .3e5 copies/uL for low-to-moderate abundance miRNAs, and around 1e7 copies/uL for high abundance miRNA) 10 19 .
  • the split guide system is specific for the desired actRNA, showing no or limited activity for 2nt mismatches, actRNA truncations, and actRNA extensions.
  • capRNAs that form stem-loops in the capture region may be sufficient to activate the RNP when bound via the anchor region, such as with capRNAs 3, 8, and most capRNAs designed for the cellular miRNA targets, resulting in high background activity (FIG. 8, FIG. 10B, and FIG. 13).
  • capRNA 1 and 2 which both have a 5nt 3’ flank and 11 nt 5’ tail but with different sequences, have dramatically different background activities (FIG. 2D).
  • the predicted structure of the capRNA bound with the split gRNA does not include any stem-loop structures that might lead to activation (FIG. 8). This suggests that the sequence of the 3’ flank and 5’ tail may play a larger role in background activation.
  • capRNAs result in varied reaction rates, consistent with the variability in activity observed in the conventional Cas13a system for different guide and target RNA combinations. This may be partially explained by differences in the stability of binding in the capture region, as it was in the anchor region.
  • the capRNA for split gRNA 2 had a slightly higher melting temperature than that for split gRNA 1 and generated a higher relative signal for the same length actRNA targets (FIG. 10E).
  • FIG. 10E we also see this trend in increased signal when the capture region length and therefore melting temperature is increased, binding more of a given actRNA.
  • capRNAs with the same anchor and capture regions but varied whether they had a 3’ flank and/or a 5’ tail (FIG. 7D). All three capRNAs had similarly low background activity, but resulted in significantly different activity when a 10nt actRNA was added. The capRNA with both the 3’ flanking sequence and 5’ tail resulted in the greatest reaction rate.
  • capRNA design will be a focus for optimizing actRNA detection with the split guide system. Designs aimed at optimizing signal- to-background ratio should consider sequence, melting temperature of the anchor and capture regions, and secondary and tertiary structure of the complex.
  • the design of the capRNA is an asset to the system, as it allows the split guide system to be tuned based on the needs of the assay. For detection with high specificity, reducing the portion of the actRNA that binds to the capRNA will increase the specificity to mismatches (FIG. 4A, FIG. 11 A). Alternately, for applications that value high sensitivity, increasing the length of the capture region will likely enhance detection at low concentrations.
  • Molecular dynamics simulations of the capRNA bound with the Cas13a and split gRNA may aid the design process, especially when these complexes are compared to predicted structures of experimentally-optimized split guide structures.
  • the Cas13a split guide system will further benefit from advances to the conventional Cas13a assay. Switching from a linear to a hairpin ssRNA reporter, for example, may reduce background signal and increase assay speed and sensitivity as it did with DNA hairpin reporters for Cas12a DNA-detection assays 25 . We could also adopt a droplet-based approach to boost sensitivity, which we have previously demonstrated in our lab for conventional Cas13a assays 5 .
  • Example 2 short RNA sequence detection with CRISPR-Casl 3a using a split guide RNA
  • Influenza A virus has a genome composed of eight viral RNA segments (Fig. 14A). Each segment is composed of an internal coding region flanked on the 5’ and 3’ end by non-coding regions (NCR). The 13nt on the 5’ end (5’ untranslated region, or UTR) and 12nt on the 3’ end (3’ UTR) are highly conserved across all segments 2425 .
  • UTR untranslated region
  • UTR capRNA designed to bind the 5’ UTR should theoretically bind all eight viral RNA (vRNA) segments (Fig. 14B).
  • one virion would yield eight detectable copies of vRNA.
  • Another approach to increasing vRNA detection is designing full-length gRNAs that target different sequences within the viral genome, which we and others have done to target SARS-CoV-2 4 ' 6 . This strategy requires testing and validation of multiple targets, and results in multiple Cas13a RNP species in one reaction. With the split gRNA system, only one split Cas13a RNP and one capRNA is needed to detect the equivalent of eight vRNA targets.
  • We tested this approach first with synthetic Influenza A viral RNA at a concentration of 7e5 copies/uL, and we found activation over background (Fig. 14C).
  • the split Cas13a RNP detected the extracted vRNA at an estimated concentration of 2e6 copies/uL.
  • This approach for detecting IAV vRNA demonstrates the ability of the split gRNA system to detect short segments that are appended to longer RNA strands; IAV vRNA can be up to ⁇ 2400nt long 25 .
  • FIG. 14 split gRNA system detects cellular miRNA and Influenza A viral RNA.
  • Influenza A is composed of eight segments of negative sense RNA. Each segment contains a coding region (light blue) and a non-coding region (NCR) on both the 5’(dark blue and pink) and 3’ ends (pink and grey). The non-coding region is composed of 5’ untranslated region (UTR) and 3’ UTR that are conserved across all eight segments. The 5’ UTR (dark blue) is 13nt and the 3’ UTR (grey) is 12 nt.
  • B Schematic illustrating the proposed method of detecting IAV vRNA with the split gRNA system.
  • the 13nt 5’ UTR (dark blue) serves at the actRNA and complements to the 13nt capture region of the capRNA. All eight segments should be detectable with the same Cas13a RNP and capRNA since the 5’ UTR is conserved.
  • C Detection of IAV vRNA with split gRNA system. Synthetic vRNA (NP segment) is added to Cas13a RNP at a final concentration of 7e5 copies/uL (blue bar, circles).
  • vRNA is extracted from Influenza A virions and added to Cas13a RNP for an estimated final concentration of 2e6 vRNA segments/uL (orange bar, rhombuses). Cas13a RNP with capRNA and no activating RNA is also shown (grey bar, empty circles). For each condition, mean reaction rate (AU/s) with three technical replicates is plotted. Stats Non-significant interactions (p>0.05) are not plotted. * is p ⁇ 0.05, ** is p ⁇ 0.01 , *** is p ⁇ 0.001 .

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Abstract

L'invention concerne des procédés et des compositions pour activer une protéine effectrice CRISPR-Cas avec un acide nucléique d'intérêt. Ceci comprend la mise en contact d'un acide nucléique de capture (capNucleicAcid) avec une protéine effectrice CRISPR-Cas et un ARN guide CRISPR-Cas en présence d'un acide nucléique d'intérêt, également dénommé acide nucléique activateur (actNucleicAcid). L'ARN guide a une séquence de guidage courte et il est dénommé ARN guide divisé. Une séquence de l'actNucleicAcid agit comme une extension de l'ARN guide.
PCT/US2025/030627 2024-05-27 2025-05-22 Compositions d'arn guide divisé et procédés d'activation d'une protéine effectrice crispr-cas avec une séquence nucléotidique courte Pending WO2025250443A1 (fr)

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