EP4240853A2 - Neuartige crispr-cas-rna-geführte endonukleasen der klasse 2 - Google Patents

Neuartige crispr-cas-rna-geführte endonukleasen der klasse 2

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Publication number
EP4240853A2
EP4240853A2 EP21815788.1A EP21815788A EP4240853A2 EP 4240853 A2 EP4240853 A2 EP 4240853A2 EP 21815788 A EP21815788 A EP 21815788A EP 4240853 A2 EP4240853 A2 EP 4240853A2
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EP
European Patent Office
Prior art keywords
sequence
type
rna
target
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21815788.1A
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English (en)
French (fr)
Inventor
Carla Alejandra GIMENEZ
Maria Julia Lara
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Science Solutions LLC
Original Assignee
Science Solutions LLC
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Publication date
Application filed by Science Solutions LLC filed Critical Science Solutions LLC
Priority claimed from PCT/US2021/057798 external-priority patent/WO2022098681A2/en
Publication of EP4240853A2 publication Critical patent/EP4240853A2/de
Pending legal-status Critical Current

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Prokaryotes have adaptive immune systems in place that utilize CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage to confer resistance to foreign genetic elements.
  • CRISPR-Cas systems act to confer adaptive immunity in bacteria and archaea via RNA-guided nucleic acid interference.
  • processed CRISPR array transcripts assemble with Cas proteincontaining surveillance complexes that recognize nucleic acids bearing sequence complementarity to the invader’ s derived segment of the crRNAs, known as the spacer.
  • Class 2 CRISPR-Cas systems are streamlined versions in which a single Cas protein (an effector endonuclease protein) bound to RNA is responsible for binding to and cleavage of a targeted sequence.
  • the programmable nature of these minimal systems has facilitated their use as a versatile technology that continues to revolutionize the field of genome manipulation.
  • compositions, pharmaceutical compositions, vectors, host cells, and kits comprising any of the proteins or polynucleotides of the engineered systems described herein.
  • compositions comprising any of the proteins or polynucleotides of the engineered systems described herein.
  • the disclosure relates to an engineered system that comprises a Class 2 CRISPR-Cas endonuclease or a nucleic acid encoding the endonuclease and a a gRNA or a nucleic acid encoding the gran.
  • the Class 2 CRISPR-Cas endonuclease can be a Class 2 Type II CRISPR-Cas endonuclease comprising at least one of the RuvC sequences of Table 7, or a sequence comprising at least 60% sequence identity thereto.
  • the Class 2 CRISPR-Cas endonuclease can be a Class 2 Type V CRISPR-Cas endonuclease comprising at least one of the RuvC sequences of Table 1, or a sequence comprising at least 60% sequence identity thereto.
  • the Class 2 CRISPR-Cas endonuclease can be a Class 2 Type VI CRISPR-Cas endonuclease comprising at least one of the HEPN sequences of Table 4, or a sequence comprising at least 60% sequence identity thereto.
  • the gRNA and the Class 2 CRISPR-Cas endonuclease generally do not naturally occur together.
  • the gRNA can be capable of hybridizing to a target sequence in a target DNA or RNA.
  • the gRNA can be capable of forming a complex with the Class 2 CRISPR-Cas endonuclease endonuclease.
  • the engineered system disclosed herein can comprise a Class 2 Type II CRISPR-Cas endonuclease; and a Class 2 Type II CRISPR-Cas gRNA.
  • the gRNA can be a single-molecule gRNA.
  • the gRNA can be a dual-molecule gRNA.
  • the endonuclease can be a Class 2 Type II CRISPR-Cas endonuclease comprising at least one of the RuvC or HNH sequences of Table 7, or a sequence comprising at least 60% sequence identity thereto or is a Class 2 Type V CRISPR-Cas endonuclease comprising at least one of the RuvC or HNH sequences of Table 1, or a sequence comprising at least 60% sequence identity thereto, and the target is target DNA.
  • the endonuclease is a Class 2 Type VI CRISPR-Cas endonuclease comprising at least one of the HEPN sequences of Table 4, or a sequence comprising at least 60% sequence identity thereto, and the target is target RNA.
  • the target RNA mRNA, tRNA, rRNA, miRNA, or siRNA.
  • the Class 2 Type II CRISPR-Cas endonuclease can comprise any one of SEQ ID NOS: 16-19, or a sequence comprising at least 60% sequence identity thereto.
  • the Class 2 Type V CRISPR-Cas endonuclease can comprises any one of SEQ ID NOS: 1-7 or 20, or a sequence comprising at least 60% sequence identity thereto.
  • the Class 2 Type VI CRISPR-Cas endonuclease can comprises any one of SEQ ID NOS: 8-15, or a sequence comprising at least 60% sequence identity thereto.
  • the disclosure relates to an engineered single-molecule gRNA that comprises a [0016] targeter-RNA comprising a spacer sequence that is capable of hybridizing with a target sequence in a target DNA; and an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded RNA duplex, the activator-RNA comprising a activator-RNA.
  • the targeter- RNA and the activator-RNA can be covalently linked to one another.
  • the single-molecule gRNA can be capable of forming a complex with a Class 2 Type II endonuclease. Hybridization of the spacer sequence to the target sequence can be capable of targeting the endonuclease to a target DNA.
  • the Class 2 Type II CRISPR-Cas endonuclease can comprise at least one of the RuvC or HNH sequences of Table 7, or a sequence comprising at least 60% sequence identity thereto.
  • the Class 2 Type II CRISPR-Cas endonuclease can comprise any one of SEQ ID NOS: 16-19, or a sequence comprising at least 60% sequence identity thereto.
  • the targeter-RNA and the activator-RNA can be arranged in a 5’ to 3’ orientation.
  • the activator-RNA and the targeter-RNA can be arranged in a 5’ to 3’ orientation.
  • the targeter-RNA and the activator-RNA can be covalently linked to one another via a linker.
  • the singlemolecule gRNA can comprise one or more sequence modifications compared to a sequence of a corresponding wild type tracrRNA and/or crRNA.
  • the targeter-RNA can comprise a spacer sequence of about 10-50 nucleotides that have 100% complementarity to a sequence in the target DNA.
  • the targeter- RNA can comprise a spacer sequence of about 10-50 nucleotides that has less than 100% complementarity to a sequence in the target DNA.
  • the method can comprise contacting the target DNA with a CRISPR-Cas endonuclease system disclosed herein.
  • the gRNA can hybridize with the target sequence, and modification of the target DNA or RNA occurs.
  • the target can be RNA.
  • the target can be mRNA, tRNA, rRNA, miRNA, or siRNA.
  • the target can be DNA.
  • the target DNA can be extrachromosomal DNA.
  • the target DNA can be part of a chromosome.
  • the target DNA can be part of a chromosome in vitro.
  • the target DNA can be part of a chromosome in vivo.
  • the target DNA or RNA can be outside a cell.
  • the target DNA or RNA can be inside a cell.
  • the target DNA or RNA can comprise a gene and/or its regulatory region.
  • the cell can be selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, a mammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell.
  • the modifying can comprise introducing a double strand break in a target DNA.
  • the contacting can occur under conditions that are permissive for non-homologous end joining or homology-directed repair.
  • the contacting can be with a target DNA to a donor polynucleotide.
  • the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA.
  • the method ma not comprise contacting the cell with a donor polynucleotide, or wherein the target DNA is modified such that nucleotides within the target DNA are deleted.
  • Disclosed herein are methods of detecting a target nucleic acid a sample the method comprising contacting the sample with a Class 2 Type V CRISPR-Cas endonuclease comprising at least one of the RuvC sequences of Table 1, or a sequence comprising at least 60% sequence identity thereto; or a Class 2 Type VI CRISPR-Cas endonuclease comprising at least one of the HEPN sequences of Table 4, or a sequence comprising at least 60% sequence identity thereto, and a gRNA comprising a spacer sequence that is capable of hybridizing with a target sequence in a target nucleic acid; and a labeled detector that does not hybridize with the spacer sequence of the gRNA; and measuring a detectable signal produced by cleavage of the labeled detector by the endonuclease, thereby detecting the target nucleic acid.
  • a Class 2 Type V CRISPR-Cas endonuclease comprising at least one of the Ru
  • the Class 2 Type V CRISPR-Cas endonuclease can comprise any one of SEQ ID NOS: 1-7 or 20, or a sequence comprising at least 60% sequence identity thereto.
  • the Class 2 Type VI CRISPR-Cas endonuclease comprises any one of SEQ ID NOS: 8-15, or a sequence comprising at least 60% sequence identity thereto.
  • the labeled detector can comprise a labeled single stranded DNA.
  • the labeled detector can comprise a labeled RNA.
  • the labeled RNA can be a single stranded RNA.
  • the labeled detector can comprise a labeled single stranded DNA/RNA chimera.
  • the labeled detector can comprise one or more modified nucleotides.
  • the target nucleic acid can be a single stranded DNA.
  • the target nucleic acid can be double stranded DNA.
  • the target nucleic acid can be single stranded RNA.
  • the target nucleic acid can be viral, plant, fungal, or bacterial.
  • the target sequence can be a sequence of a target provided in any of Tables lOa-lOf.
  • the target can be a coronvavirus.
  • the target can be a SARS- CoV-2 virus.
  • the target nucleic acid can be cDNA.
  • the target nucleic acid can be from a human cell.
  • the target nucleic acid can be from a human fetus or cancer cell.
  • the sample can comprises cells.
  • the sample can be urine, blood, serum, plasma, lymphatic fluid, cerebrospinal fluid, saliva, nasopharyngeal, oropharyngeal, nasopharyngeal/ oropharyngeal, aspirate, or biopsy sample.
  • the methods disclosed herein can comprise determining an amount of the target nucleic acid present in the sample.
  • Measuring a detectable signal can comprise one or more of: visual based detection, sensor based detection, color detection, gold nanoparticle based detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, and semiconductor-based sensing.
  • the labeled detector can comprise a modified nucleobase, a modified sugar moiety, and/or a modified nucleic acid linkage.
  • the detectable signal can be detectable in less than 15, 30, 45, 60, 90, 120, 150, 180, 210, or 240 minutes.
  • the method can further comprise an amplification step selected from loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HD A), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), Ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART), selfsustained sequence replication (3 SR), genome exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA).
  • LAMP loop-mediated isothermal amplification
  • HD A helicase-dependent amplification
  • RPA recombinase polymerase amplification
  • SDA strand displacement amplification
  • NASBA nu
  • the target nucleic acid in the sample can be present at a concentration of less than 100 pM.
  • Disclosed herein are endonucleases comprising an amino acid sequence with 30%-99.5% homology to any one of SEQ ID NOs: 1-20.
  • compositions comprising a endonucleases described herein, and optionally a pharmaceutically acceptable carrier.
  • the composition can comprise an endonucleases, optionally comprising a pharamaceutically aceptable carrier, a nucleic acid stabilizing buffer and/or or a endonuclease stabilizing buffer.
  • the endonuclease can be lyopholized, and optionally further comprises any one or more of a labeled detector, a reverse transcriptase enzyme, and reagents for loop-mediated isothermal amplification.
  • the disclosure can comrpise a recombinant expression vector comprising a DNA polynucleotide.
  • the recombinant expression vector o can comprise nucleotide sequences encoding a single endonuclease that operably linked to a promoter.
  • a host cell comprising the DNA polynucleotide.
  • a kit comprising one or more components of any of the engineered systems described herein.
  • One or more components can be lyopholized.
  • the one or more components can further comprise, a labeled reporter, and a gRNA directed to SARS-CoV-2.
  • FIG. 1 is a schematic representation of the organization of the CRISPR Cas loci around the Type V Cas t gene of the disclosure.
  • FIG. 2 shows the predicted secondary structure of the direct repeat for the Type V Cas t pre- crRNA. It is noted for this figure and all subsequent figures providing direct repeat (DR) sequences that while the sequence is provided in DNA nucleotides, it is understood that this DNA can then be transcribed into the pre-crRNA.
  • DR direct repeat
  • FIG. 3 shows the amino acid sequence of Type V Cas t (SEQ ID NO: 1) with the RuvC motifs underlined/highlighted.
  • FIG. 4 shows affinity purified Type V Cas 1’s molecular weight and purity through SDS- PAGE.
  • the arrow indicates the band containing the purified protein.
  • FIG. 5 shows a temperature-based assay to assess the stability of Type V Cas t protein.
  • FIGs. 6A-6B show ssDNA collateral cleavage of the Type V Cas t protein of the disclosures, complexed with a sgRNA for an exemplary Hantavirus target.
  • the Type V Cas t exhibits collateral activity and can cut non-target containing ssDNA.
  • FIG. 6A shows endpoint cleavage at 15, 20, 30 and 40 minutes; and
  • FIG. 6B shows the time course of cleavage. (NTC): non-target control.
  • FIG. 7 shows activity of the Type V Cas_l protein at different temperatures (25°C, 30°C, 38°C, and 50°C).
  • FIG. 8 is a schematic representation of the organization of the CRISPR Cas loci around the Type V Cas_2 gene of the disclosure.
  • FIG. 9 shows the predicted secondary structure of an auxiliary RNA and its complementarity with the direct repeat (DR) for the Type V Cas_2 pre-crRNA. Complementary regions between the DR and the auxiliary RNA are indicated in bold. Base-complementarity between the DR and the auxiliary RNA is indicated by the lines.
  • DR direct repeat
  • FIG. 10 shows the amino acid sequence of Type V Cas_2 (SEQ ID NO: 2) with the RuvC motifs underlined/highlighted.
  • FIG. 11 shows affinity purified a Type V Cas_2’s molecular weight and purity through SDS- PAGE.
  • FIG. 12 shows a temperature-based used to assay to assess the thermostability of the Type V Cas_2 protein.
  • FIG. 13 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type V Cas_3 gene of the disclosure.
  • FIG. 14 shows the predicted secondary structure of the direct repeat for the Type V Cas_3pre- crRNA.
  • FIG. 15 shows the amino acid sequence of Type V Cas_3 (SEQ ID NO: 3) with the RuvC motifs underlined/highlighted.
  • FIG. 16 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type V Cas_4 gene of the disclosure.
  • FIG. 17 shows the predicted secondary structure of the direct repeat for the Type V Cas_4 pre- crRNA.
  • FIG. 18 shows the amino acid sequence of Type V Cas_4 (SEQ ID NO: 4) with the RuvC motifs underlined/highlighted.
  • FIG. 19 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type V Cas_5 gene of the disclosure.
  • FIG. 20 shows the direct repeat sequence for the Type V Cas_5 pre-crRNA and the secondary structure of an auxiliary RNA for the Type V Cas_5. Base-complementarity between the direct repeat and the auxiliary RNA is indicated by the lines. Complementary regions between the DR and the auxiliary RNA are indicated in bold
  • FIG. 21 shows the amino acid sequence of Type V Cas_5 (SEQ ID NO: 5) with the RuvC motifs underlined/highlighted.
  • FIG. 22 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type V Cas_6 gene of the disclosure.
  • FIG. 23 shows the predicted secondary structure of an auxiliary RNA and its complementarity with the direct repeat for the pre-crRNA. Complementary regions between the DR and the auxiliary RNA are indicated in bold, and lines.
  • FIG. 24 shows the amino acid sequence of Type V Cas_6 (SEQ ID NO: 6) with the RuvC motifs underlined/highlighted.
  • FIG. 25 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type V Cas_7 gene of the disclosure.
  • FIG. 26 shows the predicted secondary structure of the direct repeat for the Type V Cas_7 pre- crRNA.
  • FIG. 27 shows the amino acid sequence of Type V Cas_7 (SEQ ID NO: 7) with the RuvC motifs underlined/highlighted.
  • FIG. 28 shows a Type V Cas_7’s molecular weight and purity through SDS-PAGE.
  • FIG. 29 shows a temperature-based assay to assess the stability of the Type V Cas_7 protein.
  • FIG. 30 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas t gene of the disclosure.
  • FIG. 31 shows the predicted secondary structure of the direct repeat for the Type VI Cas t pre-crRNA.
  • FIG. 32 shows the amino acid sequence of Type VI Cas t (SEQ ID NO: 8) with the HEPN motifs underlined/highlighted.
  • FIG. 33 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_2 gene of the disclosure.
  • FIG. 34 shows the predicted secondary structure of the direct repeat for the Type VI Cas_2 pre-crRNA.
  • FIG. 35 shows the amino acid sequence of Type VI Cas_2 (SEQ ID NO: 9) with the HEPN motifs underlined/highlighted.
  • FIG. 36 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_3 gene of the disclosure.
  • FIG. 37 shows the predicted secondary structure of the direct repeat for the Type VI Cas_3 pre-crRNA.
  • FIG. 38 shows the amino acid sequence of Type VI Cas_3 (SEQ ID NO: 10) with the HEPN motifs underlined/highlighted.
  • the HEPN motifs (E. . . .RxxxxH (SEQ ID NO: 93)) I and II are sequentially shown (highlighted in gray).
  • FIG. 39 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_4 gene of the disclosure.
  • FIG. 40 shows the predicted secondary structure of the direct repeat for the Type VI Cas_4 pre-crRNA.
  • FIG. 41 shows the amino acid sequence of Type VI Cas_4 (SEQ ID NO: 11) with the HEPN motifs underlined/highlighted.
  • FIG. 42 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_5 gene of the disclosure.
  • FIG. 43 shows the predicted secondary structure of the direct repeat for the Type VI Cas_5 pre-crRNA.
  • FIG. 44 shows the amino acid sequence of Type VI Cas_5 (SEQ ID NO: 12) with the HEPN motifs underlined/highlighted.
  • FIG. 45 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_6 gene of the disclosure.
  • FIG. 46 shows the predicted secondary structure of the direct repeat for the Type VI Cas_6 pre-crRNA.
  • FIG. 47 shows the amino acid sequence of Type VI Cas_6 (SEQ ID NO: 13).
  • the HEPN motifs (E. . . .RxxxxH (SEQ ID NO: 93)) I and II are sequentially shown (highlighted in gray).
  • FIG. 48 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_7 gene of the disclosure.
  • FIG. 49 shows the predicted secondary structure of the direct repeat for the Type VI Cas_7 pre-crRNA.
  • FIG. 50 shows the amino acid sequence of Type VI Cas_7 (SEQ ID NO: 14).
  • the HEPN motifs (E. . . .RxxxxH (SEQ ID NO: 93)) I and II are sequentially shown (highlighted in gray).
  • FIG. 51 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_8 gene of the disclosure.
  • FIG. 52 shows the predicted secondary structure of the direct repeat for the Type VI Cas_8 pre-crRNA.
  • FIG. 53 shows the amino acid sequence of Type VI Cas_8 (SEQ ID NO: 15).
  • the HEPN motifs (E. . . .RxxxxH (SEQ ID NO: 93)) I and II are sequentially shown (highlighted in gray).
  • FIG. 54 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type II Cas t gene of the disclosure.
  • FIG. 55 shows the sequence and the predicted secondary structure of the direct repeat and the tracrRNA (, and their complementary regions for the Type II Cas t .
  • FIG. 56 shows the amino acid sequence of Type II Cas t (SEQ ID NO: 16) with the RuvC motifs underlined/highlighted.
  • the RuvC I, II and III motifs are sequentially shown (highlighted in gray).
  • the conserved HNH domain is shown in italics.
  • the Campylovacterjeju Type II sequence referenced in Shmakov et al., 2015 was used as a reference for identification of the Ruv motifs.
  • FIG. 57 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type II Cas_2 gene of the disclosure.
  • FIG. 58 shows the sequence (upper part) and the predicted secondary structure (lower part) of the direct repeat and the tracrRNA, and their complementary regions for the Type II Cas_2.
  • FIG. 59 shows the amino acid sequence of Type II Cas_2 (SEQ ID NO: 17) with the RuvC motifs underlined/highlighted.
  • the RuvC I, II and III motifs are sequentially shown (highlighted in gray).
  • the conserved HNH domain is shown in italics.
  • the Campylovacterjeju Type II sequence referenced in Shmakov et al., 2015 was used as a reference for identification of the Ruv motifs.
  • FIG. 60 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type II Cas_3 gene of the disclosure.
  • FIG. 61 shows the sequence (lower part) and the predicted secondary structure (upper part) of the direct repeat and the tracrRNA, and their complementary regions for the Type II Cas_3.
  • FIG. 62 shows the amino acid sequence of Type II Cas_3 (SEQ ID NO: 18) with the RuvC motifs underlined/highlighted.
  • the RuvC I, II and III motifs are sequentially shown (highlighted in gray).
  • the conserved HNH domain is shown in italics.
  • the Campylovacterjeju Type II sequence referenced in Shmakov et al., 2015 was used as a reference for identification of the Ruv motifs.
  • FIG. 63 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type II Cas_4 gene of the disclosure.
  • FIG. 64 shows the sequence (lower part) and the predicted secondary structure (upper part) of the direct repeat and the tracrRNA (top right), and their complementary regions (top left) for the Type II Cas_4.
  • FIG. 65 shows the amino acid sequence of Type II Cas_4 (SEQ ID NO: 19) with the RuvC motifs underlined/highlighted. The RuvC I, II and III motifs are sequentially shown (highlighted in gray). The conserved HNH domain is shown in italics.
  • FIG. 66 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type V Cas_8 gene of the disclosure.
  • FIG. 67 shows the predicted secondary structure of the direct repeat for the Type V Cas_8 pre- crRNA.
  • FIG. 68 shows the amino acid sequence of Type V Cas_8 (SEQ ID NO: 20) with the RuvC motifs underlined/highlighted.
  • FIGs. 69A-69B are graphs showing colateral activity for Type V Cas t protein complexes using substrate single stranded DNA (FIG. 69A) and dsDNA (FIG. 69B) as target in the presence of magnesium or manganese as an additive.
  • FIG. 69A shows time course cleavage using a single stranded DNA target.
  • FIG. 69B shows time course cleavage using a double stranded DNA target.
  • FIGs. 70A-70B are graphs showing trans-cleavage activities of Type V Cas lprotein on single-strand DNA (FIG. 70A) and hybrid reporters but not on the single-stranded RNA tested (FIG. 70B).
  • FIG. 71 shows specific double strand DNA cleavage site of the Type V Cas_l protein.
  • FIG. 72 shows trans-cleavage activities of the Type V Cas_2protein using MnCh as additive at defined temperature range.
  • FIG. 73 shows the activity of Type V Cas_2 protein in a temperature curve (32.8°C- 45°C).
  • FIG.74 shows a graph depicting differential efficiency in dinucleotide reporter cleavage.
  • FIG. 75 shows affinity purified a Type V Cas_3’s molecular weight and purity through SDS- PAGE.
  • FIG. 76 shows a graph of a temperature-based assay to assess the stability of Type V Cas_3 protein.
  • FIGs. 77A-77D shows graphs of a Type V Cas_3. Activity test in different reaction buffer conditions.
  • FIG. 78 is a graph showing activity of the Type V Cas_3 protein at a gradient temperature, from 30°C to 50°C.
  • FIGs. 79A-79B are graphs showing DNA reporter cleavage (FIG. 79A) and RNA reporter cleavage (FIG. 79B) for Type V Cas_3.
  • FIG. 80 shows affinity purified Type V Cas_4’s molecular weight and purity through SDS- PAGE.
  • the arrow indicates the band containing the purified protein.
  • FIG. 81 shows a temperature-based assay to assess the stability of Type V Cas_4 protein.
  • FIGs. 82A-82C shows Type V Cas_4 trans-cleavage activity in three different commercial buffers, a curve of pH and different salt concentrations.
  • FIG. 83 shows the activity of Type V Cas_4 protein at different temperatures (30°C-50°C).
  • FIGs. 84A-84B are graphs showing DNA reporter cleavage (FIG. 84A) and RNA reporter cleavage (FIG. 84B) for Type V Cas_4.
  • FIG. 85 shows affinity purified Type V Cas_5’s molecular weight and purity through SDS- PAGE.
  • FIG. 86 shows a melt curve for Type V Cas_5, Type V Cas_5 with RNA guide, and protein buffer (C-).
  • FIG. 87 shows a graph of the activity test in different buffer conditions. Shows ssDNA collateral cleavage of the Type V Cas_5 protein complexed with a scoutRNA and a sgRNA of two different lengths (18 and 24 nucleotides) for an exemplary ssDNA Hantavirus target. Three buffer conditions were tested for each sgRNA.
  • FIG. 88 Shows trans-cleavage activities of the Type V Cas_5 protein in different buffer conditions at a defined temperature range.
  • FIGs. 89A-89B shows double stranded DNA (FIG. 89A) and single stranded DNA (FIG. 89B) PAM selection for Type V Cas_21_l.
  • FIG. 90 shows Type V Cas_5 trans-cleavage activity in dinucleotide single-stranded DNA reporters.
  • FIG. 91 Shows Type V Cas_5 trans-cleavage activity single-base polynucleotides singlestranded DNA reporters.
  • FIGs. 92A-92B shows ssRNA trans-cleavage activity in different buffer solutions of the Type VI Cas_2 protein complexed with a sgRNA for an exemplary ssRNA Hantavirus target.
  • FIG. 92A shows time course cleavage over 3 h.
  • FIG. 92B shows the endpoint activity after 180 min.
  • FIGs. 93A-93B shows ssRNA trans-cleavage activity of the Type VI Cas_2 protein at a defined temperature range.
  • FIG. 93 A shows time course cleavage over 3 h.
  • FIG. 93B shows the endpoint activity after 180 min.
  • FIGs. 94 shows affinity purified Type VI Cas_2’s molecular weight and purity through SDS- PAGE. The arrow indicates the band containing the purified protein.
  • FIGs. 95A-95B shows ssRNA trans-cleavage activity of the Type VI Cas_2 protein complexed with a sgRNA for an exemplary ssRNA Hantavirus target with variable flanking sequences at its 5’ and 3’ ends.
  • FIG. 96 shows the percentage of trans-cleavage activity for different ssRNA reporters of the Type VI Cas_2 protein complexed with a sgRNA for an exemplary ssRNA Hantavirus target.
  • FIGs. 97A-97B are graphs showing ssRNA and ssDNA trans-cleavage activity of the Type VI Cas_2 protein complexed with a sgRNA for an exemplary ssRNA or ssDNA Hantavirus target.
  • FIG. 97A shows time course cleavage using ssRNA target; and
  • FIG. 97B shows the time course cleavage using ssDNA target.
  • Type VI Cas Psm protein was used as control.
  • FIG. 98 shows ssRNA trans-cleavage activity in different buffer solutions of the Type VI Cas_4 protein complexed with a sgRNA for an exemplary ssRNA Hantavirus target.
  • FIG. 99 shows the trans-cleavage preference for different ssRNA reporters of the Type VI Cas_4 protein complexed with a sgRNA for an exemplary ssRNA Hantavirus target.
  • FIG. 100 shows affinity purified Type VI Cas_4’s molecular weight and purity through SDS- PAGE.
  • FIG. 101 shows ssRNA trans-cleavage activity of the Type VI Cas_4 protein at a defined temperature range.
  • FIG. 102 shows ssRNA and ssDNA trans-cleavage activity of the Type VI Cas_4 protein complexed with a sgRNA for an exemplary ssRNA or ssDNA Hantavirus target.
  • 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 enables 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
  • a sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, 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).
  • Percent complementarity and determination of percent identity or homology between particular stretches of nucleic acid sequences or 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).
  • sequence similarity or identity may be determined for an entire length of a nucleic acid or amino acid, or for an indicated portion thereof. Sequence similarity or identity may be determined using standard techniques, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48,443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheel er Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAST, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • any suitable algorithm for aligning sequences include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAST, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SO
  • WU-BLAST-2 An exemplary useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996); h t t p : / / blast. wustl/edu/blast/README.html.
  • WU-BLAST-2 uses several search parameters, which are optionally set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. Further, an additional useful algorithm is gapped BLAST as reported by Altschul et al, (1997) Nucleic Acids Res. 25, 3389-3402.
  • polypeptide and “protein” are used interchangeably herein, and refer 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.
  • 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.
  • target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • a target sequence may comprise DNA or RNA.
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence.
  • targeting sequence means the portion of a guide sequence having sufficient complemenarity with a target sequence.
  • the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Class 2 CRISPR-Cas systems generally have single-polypeptide multidomain nuclease effectors, and comprises Types II, V, and VI.
  • Class 2 Type II CRISPR-Cas endonucleases are RNA-guided DNA endonucleases (interchangeably referred to herein as Type II endonucleases, Type II endonulceases and the like).
  • Exemplary Type II endonucleases include Cas9.
  • Class 2 Type V CRISPR-Cas endonucleases are RNA-guided DNA endonucleases (interchangeably referred to herein as Type V endonucleases, Type V endonucleases and the like), and further possess collateral activity.
  • Exemplary Type V endonucleases include Casl2 (inclusive of all subtypes) and Cast 4 (inclusive of all subtypes).
  • Class 2 Type VI CRISPR-Cas endonucleases are RNA-guided RNA endonucleases (interchangeably referred to herein as Type VI endonucleases, Type VI endonulceases and the like), and further possess collateral activity.
  • Exemplary Type VI endonucleases include Cast 3 (inclusive of all subtypes).
  • Type VI endonucleases achieve RNA cleavage through conserved basic residues within its two HEPN domains.
  • the target RNA i.e. the RNA of interest, is the RNA to be targeted leading to the recruitment to, and the binding of the Type VI endonuclease at, the target site of interest on the target RNA.
  • novel Class 2 Type V CRISPR-Cas RNA-guided endonucleases and their gRNAs constituting the novel Class 2 Type V CRISPR-Cas RNA-guided systems of the disclosure.
  • engineered systems comprising: a Class 2 Type V CRISPR-Cas RNA- guided endonuclease of the disclosure and a single guide RNA, wherein the gRNA and the Class 2 Type V CRISPR-Cas RNA-guided endonuclease do not naturally occur together, wherein the gRNA is capable of hybridizing to a target sequence in a target DNA, wherein the gRNA is capable of forming a complex with the Class 2 Type V CRISPR-Cas RNA-guided endonuclease, and wherein the Class 2 Type V CRISPR-Cas RNA-guided endonuclease possesses collateral activity and is capable of collaterally cleaving a single stranded polynucleotide comprising RNA, without the use of a tracrRNA.
  • novel Type V CRISPR-Cas RNA-guided endonucleases may share certain structural, sequence, and/or functional similarities with any one of the subtypes of Casl2. In some embodiments, these endonucleases may share certain structural, sequence, and/or functional similarities with any one of the subtypes of Casl4.
  • Type V endonucleases of the are capable of cleaving target single stranded DNA (e.g. Casl4- like Type V endonucleases) and target double stranded DNA (e.g. Casl2-like Type V endonucleases).
  • Type V endonucleases additionally possess collateral activity.
  • a Type V CRISPR-Cas RNA-guided endonucleases of the disclosure comprise three RuvC motifs, responsible for catalytic activity.
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises any one of the RuvC sequences of Table 1, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises any two of the RuvC sequences of Table 1, or sequences comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises any three of the RuvC sequences of Table 1, or sequences comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a RuvC I motif selected from the group consisting of SEQ ID NO: 62, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 89, and SEQ ID NO: 135, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a RuvC II motif selected from the group consisting of SEQ ID NO: 63, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 136, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a RuvC III motif selected from the group consisting of SEQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 87, SEQ ID NO: 91, and SEQ ID NO: 137, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif selected from the group consisting of SEQ ID NO: 62, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 89, and SEQ ID NO: 135, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif selected from the group consisting of SEQ ID NO: 63, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 86, S
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif comprising the sequence of SEQ ID NO: 62, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif comprising the sequence of SEQ ID NO: 63, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and (3) a RuvC III motif comprising the sequence of SEQ
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif comprising the sequence of SEQ ID NO: 67, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif comprising the sequence of SEQ ID NO: 68, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and (3) a RuvC III motif comprising the sequence of SEQ
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif comprising the sequence of SEQ ID NO: 71, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif comprising the sequence of SEQ ID NO: 72, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and (3) a RuvC III motif comprising the sequence of SEQ ID NO: 71, or
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif comprising the sequence of SEQ ID NO: 75, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif comprising the sequence of SEQ ID NO: 76, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and (3) a RuvC III motif comprising the sequence of SEQ ID NO: 75, or a
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif comprising the sequence of SEQ ID NO: 80, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif comprising the sequence of SEQ ID NO: 81, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and (3) a RuvC III motif comprising the sequence of SEQ ID NO: 80, or a
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif comprising the sequence of SEQ ID NO: 85, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif comprising the sequence of SEQ ID NO: 86, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and (3) a RuvC III motif comprising the sequence of SEQ ID NO: 85, or a
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif comprising the sequence of SEQ ID NO: 89, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif comprising the sequence of SEQ ID NO: 90, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and (3) a RuvC III motif comprising the sequence of SEQ ID NO: 89, or
  • a Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif comprising the sequence of SEQ ID NO: 135, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif comprising the sequence of SEQ ID NO: 136, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and (3) a RuvC III motif comprising the sequence of SEQ ID NO: 135, or a
  • Table 1 provided exemplary RuvC I, RuvC II, RuvC III sequences of the Type V endonucleases of the disclosure.
  • Table 2 provides exemplary amino acid sequences for certain Type V sequences of the disclosure. Genes were identified from metagenomic samples. Scripts were run on the sequences, designed to find CRISPR sequences and accompanying genes encoding proteins showing homology with reported Cas enzymes. Comparative BlastP analyses were performed against sequences deposited in databases (NCBI, LENS), discarding those candidates showing Id% >50 with deposited proteins.
  • Presence of specific domains e.g. RuvC, HEPN
  • catalytic motifs were determined (CD-search, phmmer, UNIPROT).
  • SEQ ID NO: 1 represents a novel Type V variant of the disclosure, Type V Cas l, (1283 amino acids in length).
  • FIG. l is a schematic representation of the organization of the CRISPR Cas loci around the Type V Cas lgene of the disclosure. The loci has 60 direct repeats.
  • FIG. 3 shows the amino acid sequence of Type V Cas l (SEQ ID NO: 1) with the RuvC motifs underlined/highlighted.
  • the FnCasl2a sequence referenced in Shmakov et al., 2015 was used as a reference for identification of the Ruv motifs.
  • the RuvC I, II and III motifs are sequentially shown (highlighted in gray, with the conserved catalytic amino acids underlined).
  • FIG. 6 shows that Type V Cas l exhibits trans-cleavage activity on single-stranded DNA reporter. It is noted that
  • the Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 1 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 1 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 1 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 2 represents a novel Type V variant of the disclosure, Type V Cas_2, (1235 amino acids in length).
  • FIG. 8 is a schematic representation of the organization of the CRISPR Cas loci around the Type V Cas_2 gene of the disclosure. It is noted that the organization is similar to the casY genetic organization (referencing Chen et al. 2018, 10.3389/fmicb.2019.00928), but not identical (for example, the casl gene is split into separate open reading frames). The loci has 2 direct repeats.
  • the Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 2 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 2 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 2 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 3 represents a novel Type V variant of the disclosure, Type V Cas_3, (1259 amino acids in length).
  • FIG. 13 is a schematic representation of the organization of the CRISPR Cas cluster loci around the novel Type V Cas_3 gene of the disclosure.
  • FIG. 15 shows the amino acid sequence of Type V Cas_3 (SEQ ID NO: 3) with the RuvC motifs underlined/highlighted.
  • the FnCasl2a sequence referenced in Shmakov et al., 2015 was used as a reference for identification of the Ruv motifs.
  • the RuvC I, II and III motifs are sequentially shown (highlighted in gray, with the conserved catalytic amino acids underlined)
  • the Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 3 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 3 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 3 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 4 represents a novel Type V variant of the disclosure, Type V Cas_4, (1336 amino acids in length).
  • FIG. 16 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type V Cas_4 gene of the disclosure. The loci has 4 direct repeats.
  • FIG. 18 shows the amino acid sequence of Type V Cas_4 (SEQ ID NO: 4) with the RuvC motifs underlined/highlighted. The Fn Casl2a sequence referenced in Shmakov et al., 2015 was used as a reference for identification of the Ruv motifs. The RuvC I, II and III motifs are sequentially shown (highlighted in gray, with the conserved catalytic amino acids underlined)
  • the Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 4 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 4 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 4 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 5 represents a novel Type V variant of the disclosure, Type V Cas_5, (1146 amino acids in length).
  • FIG. 19 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type V Cas_5 gene of the disclosure.
  • FIG. 21 shows the amino acid sequence of Type V Cas_5 (SEQ ID NO: 5) with the RuvC motifs underlined/highlighted. The RuvC I, II and III motifs are sequentially shown (highlighted in gray, with the conserved catalytic amino acids underlined). The Cas sequences from Chen et al. 2019 were used as a reference to deduce the RuvC motifs.
  • the Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 5 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 5 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 5 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 6 represents a novel Type V variant of the disclosure, Type V Cas_6, (1167amino acids in length).
  • FIG. 22 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type V Cas_6 gene of the disclosure. The loci has 6 direct repeats, and a auxiliary RNA.
  • FIG. 24 shows the amino acid sequence of Type V Cas_6 (SEQ ID NO: 6) with the RuvC motifs underlined/highlighted. The RuvC I, II and III motifs are sequentially shown (highlighted in gray, with the conserved catalytic amino acids underlined). The Cas sequences from Chen et al. 2019 were used as a reference to deduce the RuvC motifs.
  • the Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 6 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 6 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 6 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 7 represents a novel Type V variant of the disclosure, Type V Cas_7, (1245 amino acids in length).
  • FIG. 25 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type V Cas_7 gene of the disclosure.
  • FIG. 27 shows the amino acid sequence of Type V Cas_7 (SEQ ID NO: 7) with the RuvC motifs underlined/highlighted. The RuvC I, II and III motifs are sequentially shown (highlighted in gray, with the conserved catalytic amino acids underlined). The FnCasl2a sequence referenced in Shmakov et al., 2015 was used as a reference for identification of the Ruv motifs.
  • the Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 7 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 7 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 7 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 20 represents a novel Type V variant of the disclosure, Type V Cas_8, (758 amino acids in length).
  • FIG. 66 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type V Cas_8 gene of the disclosure.
  • FIG. 68 shows the amino acid sequence of Type V Cas_8 (SEQ ID NO: 20) with the RuvC motifs underlined/highlighted. Probable catalytic residues are D418, E597, D696 (depicted in bold and underlined/highlighted) and D481. The RuvC I, II and III motifs are sequentially shown (highlighted in gray with the conserved catalytic amino acids underlined).
  • the Type V Cas sequences from Harrington et al. 2018 were used as reference for Ruv motifs search.
  • the Type V CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 20 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 20 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 20 and proteins with at least 30% - 99.5% sequence identity thereto.
  • Table 3 provides exemplary nucleic acid sequences for encoding certain Type V sequences of the disclosure. Also provided are exemplary codon optimized nucleic acid sequences for encoding certain Type V sequences of the disclosure, for production in E. Coli systems.
  • a Type V CRISPR-Cas RNA-guided endonuclease is encoded by a nuleic acid sequence comprising or consisting of the sequence of any one of SEQ ID NOs: 21-34 and SEQ ID NOs 59-60, or a nucleic acid sequence with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • the Type V endonuclease of the disclosure is catalytically active.
  • the Type V endonuclease of the disclosure is catalytically dead, e.g. by introducing mutations in one or more of the RuvC domains.
  • the Type V endonuclease of the disclosure targets double stranded DNA, and is a Type V nickase.
  • Type V endonucleases of the disclosure can be modified to include an aptamer.
  • Type V endonuclease of the disclosure can be further fused to domains, e.g. catalytic domains to produce dual action Cas proteins.
  • a Type V endonuclease is further fused to a base editor.
  • Type V endonucleases of the disclosure also possess collateral (trans-cleavage activity), i.e. the ability to promiscuously cleave non-targeted DNA or RNA once activated by detection of a target DNA.
  • Type V endonuclease of the disclosure is activated by a gRNA, which occurs when a sample includes a target sequence to which the gRNA hybridizes (i.e., the sample includes the targeted DNA)
  • the Type V endonuclease can become a nuclease that promiscuously cleaves oligonucleotides (e.g. ssDNAs, RNAs, chimeric RNA/DNAs) not comprising the target sequence of the gRNA (non-target oligonucleotides, to which the guide sequence of the gRNA does not hybridize).
  • oligonucleotides e.g. ssDNAs, RNAs, chimeric RNA/DNAs
  • the result can be cleavage of single stranded oligonucleotides (e.g. ssDNAs, ssRNAs, single stranded chimeric RNA/DNAs) in the sample, which can be detected using any convenient detection method (e.g., using a labeled detector DNA, RNA, or DNA/RNA chimera).
  • oligonucleotides e.g. ssDNAs, ssRNAs, single stranded chimeric RNA/DNAs
  • RNAs for Class 2 Type V CRISPR-Cas RNA-Guided Endonucleases are described in further detail below.
  • gRNAs DNA-targeting guide RNAs that direct the activities of the novel Type V endonucleases of the disclosure to a specific target sequence within a target DNA.
  • DNA-targeting RNAs are referred to herein as “gRNAs” or “gRNAs”.
  • gRNAs DNA-targeting RNAs
  • gRNAs DNA-targeting guide RNAs
  • a Type V gRNA can comprise a single segment comprising both a spacer (DNA-targeting sequence) and a Cas "protein-binding sequence" together referred to as a crRNA (e.g. Cas 12a-endonuclease).
  • a Type V gRNA can comprise a first segment (also referred to herein as a “targeter- RNA”, a “DNA-targeting segment” or a “DNA-targeting sequence”) and a second segment (also referred to herein as a “activator-RNA”, a “activator-RNA” or a "protein-binding sequence”). Also provided herein are nucleotide sequences encoding the Type V gRNAs of the disclosure. i. crRNA/Spacer Sequences for Single-RNA Guided Systems
  • Certain Type V endonucleases of the disclosure can be guided by a single crRNA (single-RNA guided systems).
  • a prototypic CRISPR-Cas protein of this class includes Casl2a.
  • the crRNA of the Type V single RNA system guides of the disclosure comprises a nucleotide sequence that is complementary to a sequence in a target DNA (DNA-targeting sequence or spacer).
  • a prototypic CRISPR-Cas protein of this class includes Cast 2a.
  • the crRNA portion of the Type V gRNAs of the disclosure can have a length of from about 25 - 50 nt. In some embodiments, the length can be about 40 - 43 nt.
  • the DNA-targeting spacer sequence of a Type V gRNA generally interacts with a target DNA in a sequence-specific manner via hybridization (i.e., base pairing). As such, the nucleotide sequence of the DNA-targeting sequence may vary and determines the location within the target DNA that the gRNA and the target DNA will interact.
  • the DNA-targeting sequence of a subject Type V gRNA can be modified (e.g., by genetic engineering) to hybridize to a desired sequence within a target DNA.
  • the DNA-targeting sequence of a subject Type V gRNA can have a length of from about 8 nucleotides to about 30 nucleotides.
  • the length can be 20-23 nucleotides.
  • the percent complementarity between the DNA-targeting spacer sequence of the crRNA and the target sequence of the target DNA can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).
  • the percent complementarity between the DNA-targeting sequence of the crRNA -RNA and the target sequence of the target DNA is 100% over the 1 - 23 contiguous 5 '-most nucleotides of the target sequence of the complementary strand of the target DNA.
  • the percent complementarity between the DNA-targeting sequence of the crRNA and the target sequence of the target DNA is at least 60% over about 1 - 23 contiguous nucleotides. In some embodiments, the percent complementarity between the DNA-targeting sequence of the crRNA and the target sequence of the target DNA is 100% over the 1 - 23 contiguous 5 '-most nucleotides of the target sequence of the complementary strand of the target DNA and as low as 0% over the remainder. In such a case, the DNA-targeting sequence can be considered to be 1 - 23 nucleotides in length.
  • a naturally unprocessed pre-crRNA of Type V comprises a direct repeat and an adjacent spacer (the portion of the crRNA that allows for targeting to a DNA molecule).
  • direct repeats partial sequence or entire sequence from unprocessed pre-crRNA are included into the Type V gRNAs of the disclosure, and improve gRNA stability.
  • Exemplary direct repeat sequences include SEQ ID NO: 61, 70, 74, and 88 (DNA sequences), or SEQ ID NOS 134, 147, 150, 151 and 153 (RNA sequences).
  • the exemplary sequences are provided in DNA nucleotides, it is understood that this DNA can then be transcribed into RNA. Accordingly the mature guides of disclosure may incorporate the entire or partial sequence of the exemplary direct repeat sequences provided herein; the guides may be composed of DNA nucleotides, analogous RNA nucleotides, or a combination of DNA and RNA nucleotides. Exemplary predicted secondary structures of the pre-crRNAs of the Type V endonucleases of the disclosure are presented in FIGS. 2, 14, 17, 26, and 67.
  • the crRNAs include non-naturally occurring, engineered direct repeat sequences.
  • the spacer sequence of a Type V gRNA of the disclosure is directed to a target sequence in a mammalian organism. In some embodiments the spacer sequence is directed to a target sequence in a non-mammalian organism.
  • the spacer sequence of a Type V gRNA of the disclosure is directed to a target sequence which is a sequence of a human.
  • the target sequence is a sequence of a non-human primate.
  • the spacer sequence of a Type V gRNA of the disclosure is directed to a target sequence in a mammalian organism, e.g. a human or non-human primate.
  • the spacer sequence of a Type V gRNA of the disclosure is directed to a target sequence in a bacteria.
  • the spacer sequence of a Type V gRNA of the disclosure is directed to a target sequence in a virus.
  • the spacer sequence of a Type V gRNA of the disclosure is directed to a target sequence in a plant.
  • Type V gRNAs of the disclosure can be modified to include an aptamer. ii. Tar eter- RNA/ Dual- RNA Guided systems
  • Type V endonucleases of the disclosure can be guided by a dual-RNA system that includes a crRNA (targeter RNA) and a auxiliary RNA; a prototypic CRISPR-Cas protein of this class includes Casl2d.
  • a dual-RNA system that includes a crRNA (targeter) and a trans-activating crRNA (tracrRNA); a prototypic CRISPR-Cas protein of this class includes Casl4.
  • the targeter-RNA of certain Type V endonulcease gRNAs of the disclosure comprise a nucleotide sequence that is complementary to a sequence in a target DNA (targeting sequence of the gRNA; DNA-targeting sequence; spacer sequence).
  • the targeter-RNA can interchangeably be referred to as a crRNA.
  • the targeter-RNA of a gRNA interacts with a target DNA in a sequence-specific manner via hybridization (i.e., base pairing).
  • the nucleotide sequence of the targeter-RNA may vary and determines the location within the target DNA that the gRNA and the target DNA will interact.
  • the targeter-RNA of a subject gRNA can be modified (e.g., by genetic engineering) to hybridize to any desired sequence within a target DNA.
  • the targeter-RNA of the Type V dual-RNA guided systems can have a length of from about 12 nucleotides to about 100 nucleotides.
  • the targeter-RNA can have a length of from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50nt, from about 12 nt to about 40 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, or from about 12 nt to about 19 nt.
  • the targeter-RNA can have a length of from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 19 nt to about 70 nt, from about 19 nt to about 80 nt, from about 19 nt to about 90 nt, from about 19 nt to about 100 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, from about 20 nt to
  • a naturally unprocessed pre-crRNA of Type V comprises a direct repeat and an adjacent spacer (the portion of the crRNA that allows for targeting to a DNA molecule).
  • direct repeats partial sequence or entire sequence
  • Exemplary direct repeat sequences include SEQ ID NO: 66, 78, and 83. It is noted that while the exemplary sequences are provided in DNA nucleotides, it is understood that this DNA can then be transcribed into RNA.
  • the mature guides of disclosure may incorporate the entire or partial sequence of the exemplary direct repeat sequences provided herein; the guides may be composed of DNA nucleotides, analogous RNA nucleotides, or a combination of DNA and RNA nucleotides.
  • Exemplary predicted secondary structures of the pre-crRNAs of the Type V endonucleases (dual RNA guided systems) of the disclosure are presented in FIGS. 9, 20, and 23.
  • the gRNAs of the disclosure include non-naturally occurring, engineered direct repeat sequences which can be incorporated into the engineered gRNAs of the disclosure. i. Spacer Sequences/Dual-RNA Guided Systems
  • gRNAs of the disclosure comprise spacer sequences, complementary to the target DNA. More specifically, the nucleotide sequence of the targeter-RNA that is complementary to a target nucleotide sequence (the DNA-targeting sequence or spacer sequence) of the target DNA can have a length at least about 12 nt.
  • the DNA- targeting sequence of the targeter-RNA that is complementary to a target sequence of the target DNA can have a length at least about 12 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt or at least about 40 nt.
  • the DNA-targeting sequence of the targeter-RNA that is complementary to a target sequence of the target DNA can have a length of from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60
  • the nucleotide sequence (the DNA-targeting sequence) of the targeter-RNA that is complementary to a nucleotide sequence (target sequence) of the target DNA can have a length at least about 12 nt. In some embodiments, the DNA-targeting sequence of the targeter-RNA that is complementary to a target sequence of the target DNA is 20 nucleotides in length. In some embodiments, the DNA-targeting sequence of the targeter-RNA that is complementary to a target sequence of the target DNA is 19 nucleotides in length.
  • the percent complementarity between the spacer sequence of the targeter-RNA and the target sequence of the target DNA can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).
  • the percent complementarity between the DNA-targeting sequence of the targeter- RNA and the target sequence of the target DNA is 100% over the 1-25 contiguous 5 '-most nucleotides of the target sequence of the complementary strand of the target DNA.
  • the percent complementarity between the DNA-targeting sequence of the targeter-RNA and the target sequence of the target DNA is at least 60% over about 1 - 25 contiguous nucleotides. In some embodiments, the percent complementarity between the DNA-targeting sequence of the targeter-RNA and the target sequence of the target DNA is 100% over the 1 - 25 contiguous 5 '-most nucleotides of the target sequence of the complementary strand of the target DNA and as low as 0% over the remainder. In such a case, the DNA-targeting sequence can be considered to be 1 - 25 nucleotides in length.
  • the spacer sequence of a Type V dual-RNA guided system of the disclosure is directed to a target sequence in a mammalian organism. In some embodiments the spacer sequence is directed to a target sequence in a non-mammalian organism.
  • the spacer sequence of a Type V dual-RNA guided system of the disclosure is directed to a target sequence which is a sequence of a human.
  • the target sequence is a sequence of a non-human primate.
  • the spacer sequence of a Type V dual-RNA guided system of the disclosure is directed to a target sequence selected of a therapeutic target.
  • the spacer sequence of a Type V dual-RNA guided system of the disclosure is directed to a target sequence selected of a diagnostic target - for example in such embodiments a labeled catalytically dead Type II endonuclease of the disclosure and a gRNA directed to a diagnostic target DNA is contacted with the target DNA, or a cell comprising the target DNA, or a sample comprising the target DNA.
  • the activator-RNA of certain Type V gRNA of the disclosure binds with its cognate Type V endonuclease of the disclosure (e.g. Type V Cas_8 of the disclosure).
  • the activator-RNA can interchangeably be referred to as a tracrRNA.
  • the gRNA guides the bound Type V endonuclease to a specific nucleotide sequence within target DNA via the above described targeter-RNA.
  • the activator- RNA of a Type V gRNA comprises two stretches of nucleotides that are complementary to one another.
  • dual molecule (two-molecule) Type V gRNAs for the novel Type V endonucleases of the disclosure.
  • Such gRNAs comprise two separate RNA molecules (tracRNA or auxialiary RNA; and the targeting RNA- crRNA).
  • Each of the two RNA molecules of a subject double-molecule gRNA comprises a stretch of nucleotides that are complementary to one another such that the complementary nucleotides of the two RNA molecules hybridize to form the double stranded RNA duplex of the gRNA.
  • a dual-molecule gRNA can be designed to allow for controlled (i.e., conditional) binding of a targeter-RNA with an activator-RNA.
  • a dual-molecule gRNA is not functional unless both the activator-RNA and the targeter-RNA are bound in a functional complex with Type V endonulceases of the disclosure, a dual-molecule gRNA can be inducible (e.g., drug inducible) by rendering the binding between the activator-RNA and the targeter-RNA to be inducible.
  • RNA aptamers can be used to regulate (i.e., control) the binding of the activator-RNA with the targeter-RNA.
  • the activator-RNA and/or the targeter-RNA can comprise an RNA aptamer sequence.
  • the dual-molecule guide can be modified to include an aptamer.
  • an aptamer iv. Engineered Single-molecule Type V Endonulcease gRNAs
  • Type V gRNAs that comprises a singlemolecule gRNA (interchangeably referred to herein as a sgRNA), for the novel Type V endonucleases of the disclosure.
  • an engineered single-molecule gRNA comprising: a. a targeter-RNA that is capable of hybridizing with a target sequence in a target DNA; and b. an activator-RNA that is capable of hybridizing with the targeter-RNA to form a doublestranded RNA duplex, the activator-RNA comprising a activator-RNA, wherein the targeter-RNA and the activator-RNA are covalently linked to one another, wherein the single-molecule gRNA is capable of forming a complex with a novel Type V endonuclease of the disclosure, and wherein hybridization of the targeter-RNA to the target sequence is capable of targeting the Type V endonuclease of the disclosure to the target DNA.
  • a subject engineered single-molecule gRNA comprises two segments of nucleotides (a targeter-RNA and an activator-RNA) that are complementary to one another, can be covalently linked by intervening nucleotides ("linkers” or “linker nucleotides”), and hybridize to form the double stranded RNA duplex (dsRNA duplex) of the activator-RNA, whereby resulting in a stem-loop structure.
  • the targeter-RNA and the activator-RNA are covalently linked via the 3' end of the targeter-RNA and the 5' end of the activator-RNA.
  • the activator-RNA is covalently linked via the 5' end of the targeter-RNA and the 3' end of the activator-RNA.
  • the targeter-RNA and the activator-RNA are arranged in a 5’ to 3’ orientation.
  • the activator-RNA and the targeter-RNA are arranged in a 5’ to 3’ orientation.
  • the single molecule gRNA comprises one or more sequence modifications compared to a sequence of a corresponding wild type tracrRNA and/or crRNA.
  • the targeter-RNA and the activator-RNA are covalently linked to one another via a linker.
  • the linker of a single-molecule gRNA can have a length of from about 3 nucleotides to about 30 nucleotides. In exemplary embodiments, the linker of a single- molecule gRNA is 4, 5, 6, or 7 nt.
  • An exemplary single-molecule gRNA comprises two complementary stretches of nucleotides that hybridize to form a dsRNA duplex.
  • one of the two complementary stretches of nucleotides of the single -molecule gRNA (or the DNA encoding the stretch) is at least about 60% identical to one of the activator-RNA.
  • one of the two complementary stretches of nucleotides of the single-molecule gRNA is at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical or 100 % identical to an activator-RNA.
  • the activator-RNA and targeter-RNA segments can be engineered, while ensuring that the structure of the protein-binding domain of the gRNA is conserved.
  • RNA folding structure of a naturally occurring protein-binding domain of a DNA-targeting RNA can be taken into account in order to design artificial protein- binding domains (either dual-molecule or single-molecule versions).
  • the activator-RNA in a single-molecule gRNA can have a length of from about 10 nucleotides to about 100 nucleotides.
  • the activator-RNA can have a length of from about 15 nucleotides (nt) to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt or from about 15 nt to about 25 nt.
  • the dsRNA duplex of the activator-RNA can have a length from about 6 nucleotides (nt) to about 50bp.
  • the dsRNA duplex of the activator-RNA can have a length from about 6 nt to about 40 nt, from about 6 nt to about 30bp, from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8 nt to about 30bp, from about 8 nt to about 25 nt, from about 8 nt to about 20 nt or from about 8 nt to about 15 nt.
  • the dsRNA duplex of the activator-RNA can have a length from about from about 8 nt to about 10 nt, from about 10 nt to about 15 nt, from about 15 nt to about 18 nt, from about 18 nt to about 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt, from about 35 nt to about 40 nt, or from about 40 nt to about 50 nt.
  • the dsRNA duplex of the activator- RNA has a length of 8 - 15 base pairs.
  • the percent complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the activator-RNA can be at least about 60%.
  • the percent complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the activator-RNA can be at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% .
  • the percent complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the activator-RNA is 100%.
  • the spacer sequence of a Type V gRNA (whether it is a single molecule gRNA or a dual molecule gRNA) of the disclosure is directed to a target sequence in a mammalian organism, e.g. a human or non-human primate. In some embodiments, the spacer sequence of a Type V gRNA of the disclosure is directed to a target sequence in a bacteria.
  • the spacer sequence of a Type V gRNA of the disclosure is directed to a target sequence in a virus. In some embodiments, the spacer sequence of a Type V gRNA of the disclosure is directed to a target sequence in a plant.
  • the single-molecule Type V gRNAs of the disclosure can be modified to include an aptamer.
  • v. gRNA Arrays v. gRNA Arrays
  • the Type V gRNAs of the disclosure can be provided as gRNA arrays.
  • Such gRNA arrays of the disclosure include more than one gRNA arrayed in tandem, and can be processed into two or more individual gRNAs.
  • a precursor Type V gRNA array comprises two or more (e.g., 3 or more, 4 or more, 5 or more, 2, 3, 4, or 5) gRNAs (e.g., arrayed in tandem as precursor molecules).
  • two or more gRNAs can be present on an array (a precursor gRNA array).
  • a Type V endonuclease of the disclosure can cleave the precursor gRNA array into individual gRNAs.
  • a Type V gRNA array includes 2 or more gRNAs (e.g., 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, gRNAs).
  • the gRNAs of a given array can target (i.e., can include guide sequences that hybridize to) different target sites of the same target DNA.
  • two or more gRNAs of a precursor gRNA array have the same guide sequence.
  • the precursor gRNA array comprises two or more gRNAs that target different target sites within the same target DNA.
  • the precursor gRNA array comprises two or more gRNAs that target different target DNAs.
  • gRNA guide RNA
  • systems comprising (a) Type VI endonuclease, or a nucleic acid encoding the Type VI endonuclease; and (b) a Type VI gRNA, or a nucleic acid encoding the Type VI gRNA, wherein the gRNA and the Type VI endonuclease do not naturally occur together, wherein the gRNA is capable of hybridizing to a target sequence in a target single stranded RNA, and the gRNA is capable of forming a complex with the Type VI endonuclease.
  • Type VI CRISPR-Cas RNA-guided endonucleases may share certain structural, sequence, and/or functional similarities with any one of the subtypes of Casl3 (e.g. Casl3a, Casl3b).
  • Type VI endonucleases are useful for RNA targeting and modification.
  • Type VI targets ssRNA and requires a protospacer flanking sequence (PFS) instead of the PAM required for dsDNA unwinding, e.g. for Type II and Type V endonucleases.
  • PPS protospacer flanking sequence
  • a Type VI CRISPR-Cas RNA-guided endonucleases of the disclosure comprise two HEPN motifs, generally of the motif E RXXXXH (SEQ ID NO: 93), also referred to as E. . . .R-X4-H (SEQ ID NO: 93).
  • E RXXXXH SEQ ID NO: 93
  • R-X4-H SEQ ID NO: 93
  • a Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises any one of the HEPN sequences of Table 4, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises any two of the HEPN sequences of Table 4, or sequences comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a HEPN motif selected from the group consisting of SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 113, and SEQ ID NO: 197, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a first HEPN motif selected from the group consisting of SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 113, and SEQ ID NO: 197, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and comprises a second HEPN motif selected from the group consisting of
  • a Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a first HEPN motif comprising the amino acid sequence of SEQ ID NO: 94, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and comprises a second HEPN motif comprising the amino acid sequence of SEQ ID NO: 95 or SEQ ID NO: 197, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a first HEPN motif comprising the amino acid sequence of SEQ ID NO: 97, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and comprises a second HEPN motif comprising the amino acid sequence of SEQ ID NO: 95 or SEQ ID NO: 197, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a first HEPN motif comprising the amino acid sequence of SEQ ID NO: 99, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and comprises a second HEPN motif comprising the amino acid sequence of SEQ ID NO: 100, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a first HEPN motif comprising the amino acid sequence of SEQ ID NO: 95 or SEQ ID NO: 197, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and comprises a second HEPN motif comprising the amino acid sequence of SEQ ID NO: 102, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a first HEPN motif comprising the amino acid sequence of SEQ ID NO: 104, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and comprises a second HEPN motif comprising the amino acid sequence of SEQ ID NO: 105, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a first HEPN motif comprising the amino acid sequence of SEQ ID NO: 107, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and comprises a second HEPN motif comprising the amino acid sequence of SEQ ID NO: 108, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a first HEPN motif comprising the amino acid sequence of SEQ ID NO: 110 or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and comprises a second HEPN motif comprising the amino acid sequence of SEQ ID NO: 111, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a first HEPN motif comprising the amino acid sequence of SEQ ID NO: 99, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and comprises a second HEPN motif comprising the amino acid sequence of SEQ ID NO: 113, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • Table 4 provided exemplary HEPN sequences of the Type VI endonucleases of the disclosure.
  • Table 5 provides exemplary amino acid sequences for certain Type VI sequences of the disclosure. Genes were identified from metagenomic samples. Scripts were run on the sequences, designed to find CRISPR sequences and accompanying genes encoding proteins showing homology with reported Cas enzymes. Comparative BlastP analyses were performed against sequences deposited in databases (NCBI, LENS), discarding those candidates showing Id% >50 with deposited proteins. Presence of specific domains (e.g. RuvC, HEPN) and catalytic motifs were determined (CD-search, phmmer, UNIPROT).
  • specific domains e.g. RuvC, HEPN
  • catalytic motifs were determined (CD-search, phmmer, UNIPROT).
  • SEQ ID NO: 8 represents a novel Type VI variant of the disclosure, Type VI Cas l, (1148 amino acids in length).
  • FIG. 30 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas l gene of the disclosure.
  • FIG. 32 shows the amino acid sequence of Type VI Cas l (SEQ ID NO: 8) with the HEPN motifs underlined/highlighted. The HEPN motifs (E. . . .RxxxxH (SEQ ID NO: 93)) I and II are sequentially shown (highlighted in gray).
  • the Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 8 and proteins with at least 30% - 99.5% sequence identity thereto. Accordingly, provided herein are proteins comprising the amino acid sequence of SEQ ID NO: 8 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% or at least 99.5% sequence identity thereto.
  • SEQ ID NO: 9 represents a novel Type VI variant of the disclosure, Type VI Cas_2, (1138 amino acids in length).
  • FIG. 33 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_2 gene of the disclosure.
  • FIG. 35 shows the amino acid sequence of Type VI Cas_2 (SEQ ID NO: 9) with the HEPN motifs underlined/highlighted. The HEPN motifs (E. . . .RxxxxH (SEQ ID NO: 93)) I and II are sequentially shown (highlighted in gray).
  • the Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 9 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 9 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 9 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 10 represents a novel Type VI variant of the disclosure, Type VI Cas_3, (1093 amino acids in length).
  • FIG. 36 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_3 gene of the disclosure.
  • FIG. 38 shows the amino acid sequence of Type VI Cas_3 (SEQ ID NO: 10) with the HEPN motifs underlined/highlighted. The HEPN motifs (E. . . .RxxxxH (SEQ ID NO: 93)) I and II are sequentially shown (highlighted in gray).
  • the Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 10 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 10 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 10 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 11 represents a novel Type VI variant of the disclosure, Type VI Cas_4, (1236 amino acids in length).
  • FIG. 39 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_4 gene of the disclosure.
  • FIG. 41 shows the amino acid sequence of Type VI Cas_4 (SEQ ID NO: 11) with the HEPN motifs underlined/highlighted. The HEPN motifs (E. . . .RxxxxH (SEQ ID NO: 93)) I and II are sequentially shown (highlighted in gray).
  • the Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 11 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 11 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 11 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 12 represents a novel Type VI variant of the disclosure, Type VI Cas_5, (1092 amino acids in length).
  • FIG. 42 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_5 gene of the disclosure.
  • FIG. 44 shows the amino acid sequence of Type VI Cas_5 (SEQ ID NO: 12) with the HEPN motifs underlined/highlighted.
  • the (E. . . .CNxxxH (SEQ ID NO: 142)) motif was previously observed aligned with HEPN motif (Anantharaman et al. Biology Direct 2013, 8: 15).
  • the HEPN (E. . . RxxxxH (SEQ ID NO: 93)) and (E. . . CNxxxH (SEQ ID NO: 142)) motifs are shown in gray.
  • the Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 12 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 12 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 12 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 13 represents a novel Type VI variant of the disclosure, Type VI Cas_6, (1053 amino acids in length).
  • FIG. 45 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_6 gene of the disclosure.
  • FIG. 47 shows the amino acid sequence of Type VI Cas_6 (SEQ ID NO: 13).
  • the HEPN motifs E. . . RxxxxH (SEQ ID NO: 93)
  • I and II are sequentially shown (highlighted in gray).
  • the Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 13 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 13 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 13 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 14 represents a novel Type VI variant of the disclosure, Type VI Cas_7, (1163 amino acids in length).
  • FIG. 48 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_7 gene of the disclosure.
  • FIG. 50 shows the amino acid sequence of Type VI Cas_7 (SEQ ID NO: 14).
  • the HEPN motifs E. . . RxxxxH (SEQ ID NO: 93)
  • I and II are sequentially shown (highlighted in gray).
  • the Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 14 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 14 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 14 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 15 represents a novel Type VI variant of the disclosure, Type VI Cas_8, (1124 amino acids in length).
  • FIG. 51 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type VI Cas_8 gene of the disclosure.
  • FIG. 53 shows the amino acid sequence of Type VI Cas_8 (SEQ ID NO: 15).
  • the HEPN motifs E. . . RxxxxH (SEQ ID NO: 93)
  • I and II are sequentially shown (highlighted in gray).
  • the Type VI CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 15 and proteins with at least 30% - 99.5% sequence identity thereto. Accordingly, provided herein are proteins comprising the amino acid sequence of SEQ ID NO: 15 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 15 and proteins with at least 30% - 99.5% sequence identity thereto.
  • Table 6 provides exemplary nucleic acid sequences for encoding certain Type VI sequences of the disclosure.
  • a Type VI CRISPR-Cas RNA-guided endonuclease is encoded by a nucleic acid sequence comprising or consisting of the sequence of any one of SEQ ID NOs: 35-50, or a nucleic acid sequence with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • the Type VI endonuclease of the disclosure is catalytically active.
  • the Type VI endonuclease of the disclosure is catalytically dead, e.g. by introducing mutations in one or both of the HEPN domains.
  • Type VI endonucleases of the disclosure can be modified to include an aptamer.
  • Type VI endonuclease of the disclosure can be further fused to domains, e.g. catalytic domains to produce dual action Cas proteins.
  • a Type VI endonuclease is further fused to a base editor.
  • the Type VI endonucleases of the disclosure also possess collateral (trans-cleavage activity), i.e. the ability to promiscuously cleave non-targeted DNA or RNA once activated by detection of a target DNA.
  • Type VI endonuclease of the disclosure is activated by the binding of a gRNA, which occurs when a sample includes a target sequence to which the gRNA hybridizes (i.e., the sample includes the targeted ssRNA)
  • the Type VI endonuclease can become a nuclease that promiscuously cleaves oligonucleotides (ssRNAs) not comprising the target sequence of the gRNA (non-target oligonucleotides, to which the guide sequence of the gRNA does not hybridize).
  • the result can be cleavage of single stranded reporter oligonucleotides (e.g. labeled) in the sample, which can be detected using any convenient detection method.
  • a target RNA e.g., in some embodiments above a threshold amount
  • methods and compositions for detecting a target RNA in a sample e.g., in some embodiments above a threshold amount
  • methods and compositions for cleaving non-target RNA oligonucleotides which can be utilized as detectors. These embodiments are described in further detail below.
  • RNA-targeting RNAs that direct the activities of the novel Type VI endonucleases of the disclosure to a specific target sequence within a target ssRNA.
  • RNA-targeting RNAs are also referred to herein as “gRNAs” or “gRNAs”
  • gRNAs RNA-targeting RNAs
  • a Type VI gRNA comprises a single segment comprising both a spacer (DNA-targeting sequence) and a Type VI "protein-binding sequence" together referred to as a crRNA.
  • nucleotide sequences encoding the Type VI gRNAs of the disclosure are also provided herein. i. Spacer Sequences
  • the Type VI endonucleases of the disclosure are single crRNA-guided endonucleases (single guide RNA, sgRNA, while the Type II endonucleases of the disclosure are guided by a dual-RNA system consisting of a crRNA and a trans-activating crRNA (tracrRNA).
  • the crRNA of the Type VI guides of the disclosure comprises a nucleotide sequence that is complementary to a sequence in a target RNA.
  • the crRNA portion of the Type VI gRNAs of the disclosure can have a length of from about 45 to about 70 nt. In some embodiments, the length can be about 60 to about 65 nt.
  • the RNA-targeting spacer sequence of a Type VI gRNA generally interacts with a target RNA in a sequence-specific manner via hybridization (i.e., base pairing). As such, the nucleotide sequence of the RNA-targeting sequence may vary and determines the location within the target RNA that the gRNA and the target RNA will interact.
  • the RNA-targeting sequence of a subject Type VI gRNA can be modified (e.g., by genetic engineering) to hybridize to a desired sequence within a target RNA.
  • the RNA-targeting sequence of a subject Type VI gRNA can have a length of from about 18 nucleotides to about 30 nucleotides.
  • the length can be 27 nucleotides.
  • the percent complementarity between the RNA-targeting spacer sequence of the crRNA and the target sequence of the target RNA can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).
  • the percent complementarity between the RNA-targeting sequence of the crRNA -RNA and the target sequence of the target RNA is 100% over the 1 - 27 contiguous 5 '-most nucleotides of the target sequence of the complementary strand of the target RNA.
  • the percent complementarity between the RNA-targeting sequence of the crRNA and the target sequence of the target RNA is at least 60% over about 1 - 27 contiguous nucleotides. In some embodiments, the percent complementarity between the RNA-targeting sequence of the crRNA and the target sequence of the target RNA is 100% over the 1 - 27 contiguous 5 '-most nucleotides of the target sequence of the complementary strand of the target RNA and as low as 0% over the remainder. In such a case, the RNA-targeting sequence can be considered to be 1 - 27 nucleotides in length.
  • a naturally unprocessed pre-crRNA of Type VI comprises a direct repeat and an adjacent spacer (the portion of the crRNA that allows for targeting to a RNA molecule).
  • direct repeats partial sequence or entire sequence
  • Exemplary direct repeat sequences include SEQ ID NO: 92, 96, 98, 101, 103, 106, 109, and 112 (DNA sequences) or SEQ ID NOS 154-161 (RNA sequences). It is noted that while the exemplary sequences are provided in DNA nucleotides, it is understood that this DNA can then be transcribed into RNA.
  • the mature guides of disclosure may incorporate the entire or partial sequence of the exemplary direct repeat sequences provided herein; the guides may be composed of DNA nucleotides, analogous RNA nucleotides, or a combination of DNA and RNA nucleotides.
  • Exemplary predicted secondary structures of the pre-crRNAs of the Type VI endonucleases of the disclosure are presented in FIGS. 31, 34, 37, 40, 43, 46, 49, and 52.
  • the crRNAs include non-naturally occurring, engineered direct repeat sequences.
  • the spacer sequence of a Type VI gRNA of the disclosure is directed to a target sequence in a mammalian organism. In some embodiments the spacer sequence is directed to a target sequence in a non-mammalian organism.
  • the spacer sequence of a Type VI gRNA of the disclosure is directed to a target sequence which is a sequence of a human.
  • the target sequence is a sequence of a non -human primate.
  • the spacer sequence of a Type VI gRNA of the disclosure is directed to a target sequence in a mammalian organism, e.g. a human or non-human primate.
  • the spacer sequence of a Type VI gRNA of the disclosure is directed to a target sequence in a bacteria.
  • the spacer sequence of a Type VI gRNA of the disclosure is directed to a target sequence in a virus.
  • the spacer sequence of a Type VI gRNA of the disclosure is directed to a target sequence in a plant.
  • Type VI gRNAs of the disclosure can be modified to include an aptamer. ii. gRNA Arrays
  • the Type VI gRNAs of the disclosure can be provided as gRNA arrays.
  • Such gRNA arrays of the disclosure include more than one gRNA arrayed in tandem, and can be processed into two or more individual gRNAs.
  • a precursor Type VI gRNA array comprises two or more (e.g., 3 or more, 4 or more, 5 or more, 2, 3, 4, or 5) gRNAs (e.g., arrayed in tandem as precursor molecules).
  • two or more gRNAs can be present on an array (a precursor gRNA array).
  • a Type VI endonuclease of the disclosure can cleave the precursor gRNA array into individual gRNAs.
  • a Type VI gRNA array includes 2 or more gRNAs (e.g., 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, gRNAs).
  • the gRNAs of a given array can target (i.e., can include guide sequences that hybridize to) different target sites of the same target RNA.
  • two or more gRNAs of a precursor gRNA array have the same guide sequence.
  • the precursor gRNA array comprises two or more gRNAs that target different target sites within the same target RNA.
  • the precursor gRNA array comprises two or more gRNAs that target different target RNAs.
  • a gRNA may comprise only RNA nucleotides, may comprise RNA and DNA nucleotides, or may comprise only DNA nucleotides, and thus while referred to as a gRNA, may comprise non RNA-nucleotides.
  • systems comprising (a) a Type II endonuclease, or a nucleic acid encoding the Type II endonuclease; and (b) a Type II gRNA, or a nucleic acid encoding the Type II gRNA, wherein the gRNA and the Type II endonuclease do not naturally occur together, wherein the gRNA is capable of hybridizing to a target sequence in a target DNA, and the gRNA is capable of forming a complex with the Type II endonuclease.
  • a Type II endonuclease or a nucleic acid encoding the Type II endonuclease
  • gRNA and the Type II endonuclease do not naturally occur together, wherein the gRNA is capable of hybridizing to a target sequence in a target DNA, and the gRNA is capable of forming a complex with the Type II endonuclease.
  • Type II CRISPR-Cas RNA-guided endonucleases may share certain structural, sequence, and/or functional similarities with any one of the subtypes of Cas9.
  • a Type II CRISPR-Cas RNA-guided endonucleases of the disclosure comprise three RuvC motifs and a HNH domain, responsible for catalytic activity.
  • a Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises any one of the RuvC sequences of Table 7, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises any two of the RuvC sequences of Table 7, or sequences comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises any three of the RuvC sequences of Table 7, or sequences comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a HNH domain selected from the group consisting of SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 141, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a RuvC I motif selected from the group consisting of SEQ ID NO: 116, SEQ ID NO: 121, SEQ ID NO: 126, and SEQ ID NO: 131, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a RuvC II motif selected from the group consisting of SEQ ID NO: 117, SEQ ID NO: 122, SEQ ID NO: 127, and SEQ ID NO: 132, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a RuvC III motif selected from the group consisting of SEQ ID NO: 118, SEQ ID NO: 123, SEQ ID NO: 128, and SEQ ID NO: 133, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif selected from the group consisting of of SEQ ID NO: 116, SEQ ID NO: 121, SEQ ID NO: 126, and SEQ ID NO: 131, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif selected from the group consisting of SEQ ID NO: 117, SEQ ID NO: 122, SEQ ID NO: 127, and SEQ ID NO: 132, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
  • the Type II CRISPR-Cas RNA- guided endonuclease may further comprise a HNH domain selected from the group consisting of SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 141, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif comprising the sequence of SEQ ID NO: 116, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif comprising the sequence of SEQ ID NO: 117, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and (3) a RuvC III motif comprising the sequence of SEQ
  • the Type II CRISPR-Cas RNA- guided endonuclease may further comprise a HNH domain selected from the group consisting of SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 141, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • the HNH domain comprises the sequence of SEQ ID NO: 138, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif comprising the sequence of SEQ ID NO: 121, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif comprising the sequence of SEQ ID NO: 122, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and (3) a RuvC III motif comprising the sequence of SEQ
  • the Type II CRISPR-Cas RNA- guided endonuclease may further comprise a HNH domain selected from the group consisting of SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 141, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • the HNH domain comprises the sequence of SEQ ID NO: 139, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif comprising the sequence of SEQ ID NO: 126, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif comprising the sequence of SEQ ID NO: 127, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and (3) a RuvC III motif comprising the sequence of SEQ
  • the Type II CRISPR-Cas RNA- guided endonuclease may further comprise a HNH domain selected from the group consisting of SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 141, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • the HNH domain comprises the sequence of SEQ ID NO: 140, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • a Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises a (1) RuvC I motif comprising the sequence of SEQ ID NO: 131, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; (2) a RuvC II motif comprising the sequence of SEQ ID NO: 132, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto; and (3) a RuvC III motif comprising the sequence of SEQ
  • the Type II CRISPR-Cas RNA- guided endonuclease may further comprise a HNH domain selected from the group consisting of SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 141, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • the HNH domain comprises the sequence of SEQ ID NO: 141, or a sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • Table 7 provided exemplary RuvC I, RuvC II, RuvC III, and HNH domain sequences of the Type II endonucleases of the disclosure.
  • Table 8 shows exemplary amino acid sequences for novel Type II sequences of the disclosure.
  • Genes were identified from metagenomic samples. Scripts were run on the sequences, designed to find CRISPR sequences and accompanying genes encoding proteins showing homology with reported Cas enzymes. Comparative BlastP analyses were performed against sequences deposited in databases (NCBI, LENS), discarding those candidates showing Id% >50 with deposited proteins. Presence of specific domains (e.g. RuvC, HEPN) and catalytic motifs were determined (CD-search, phmmer, UNIPROT).
  • specific domains e.g. RuvC, HEPN
  • catalytic motifs were determined (CD-search, phmmer, UNIPROT).
  • SEQ ID NO: 16 represents a novel Type II variant of the disclosure, Type II Cas l, (1091 amino acids in length
  • FIG. 54 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type II Cas l gene of the disclosure.
  • FIG. 56 shows the amino acid sequence of Type II Cas l (SEQ ID NO: 16) with the RuvC motifs underlined/highlighted. The RuvC I, II and III motifs are sequentially shown (highlighted in gray). The HNH domain is shown in italics. The Campylovacterjeju Type II sequence referenced in Shmakov et al., 2015 was used as a reference for identification of the Ruv motifs.
  • the Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 16 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 16 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 16 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 17 represents a novel Type II variant of the disclosure, Type II Cas_2, (1565 amino acids in length).
  • FIG. 57 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type II Cas_2 gene of the disclosure. There are two putative tracRNA (tracRNAl, tracRNA2). Likely only one has sufficient complementarity to enable stable interaction.
  • FIG. 59 shows the amino acid sequence of Type II Cas_2 (SEQ ID NO: 17) with the RuvC motifs underlined/highlighted. The RuvC I, II and III motifs are sequentially shown (highlighted in gray). The HNH domain is shown in italics. The Campylovacterjeju Type II sequence referenced in Shmakov et al., 2015 was used as a reference for identification of the Ruv motifs.
  • the Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 17 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 17 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 17 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 18 represents a novel Type II variant of the disclosure, Type II Cas_3, (1064 amino acids in length).
  • FIG. 60 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type II Cas_3 gene of the disclosure.
  • FIG. 62 shows the amino acid sequence of Type II Cas_3 (SEQ ID NO: 18) with the RuvC motifs underlined/highlighted. The RuvC I, II and III motifs are sequentially shown (highlighted in gray). The HNH domain is shown in italics. The Campylovacterjeju Type II sequence referenced in Shmakov et al., 2015 was used as a reference for identification of the Ruv motifs.
  • the Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 18 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 18 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 18 and proteins with at least 30% - 99.5% sequence identity thereto.
  • SEQ ID NO: 19 represents a novel Type II variant of the disclosure, Type II Cas_4, (1024 amino acids in length).
  • FIG. 63 is a schematic representation of the organization of the CRISPR Cas cluster loci around the Type II Cas_4 gene of the disclosure.
  • FIG. 65 shows the amino acid sequence of Type II Cas_4 (SEQ ID NO: 19) with the RuvC motifs underlined/highlighted. The RuvC I, II and III motifs are sequentially shown (highlighted in gray). The HNH domain is shown in italics. The Campylovacterjeju Type II sequence referenced in Shmakov et al., 2015 was used as a reference for identification of the Ruv motifs.
  • the Type II CRISPR-Cas RNA-guided endonuclease of the disclosure comprises the amino acid sequence of SEQ ID NO: 19 and proteins with at least 30% - 99.5% sequence identity thereto.
  • proteins comprising the amino acid sequence of SEQ ID NO: 19 and proteins with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99.5% sequence identity thereto.
  • nucleic acids encoding the proteins comprising the amino acid sequence of SEQ ID NO: 19 and proteins with at least 30% - 99.5% sequence identity thereto.
  • Table 9 provides exemplary nucleic acid sequences for encoding certain Type II sequences of the disclosure. Also provided are exemplary E. coli codon optimized nucleic acid sequences for encoding certain Type II sequences of the disclosure.
  • a Type II CRISPR-Cas RNA-guided endonucleases of the disclosure is encoded by a nucleic acid sequence comprising or consisting of the sequence of any one of SEQ ID NOs: 51-58, or a nucleic acid sequence with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least
  • the Type II endonuclease of the disclosure is catalytically active.
  • the Type II endonuclease of the disclosure is catalytically dead e.g. by introducing mutations in one or more of the RuvC domains.
  • the Type II endonuclease of the disclosure is a Type II nickase.
  • Type II endonucleases of the disclosure can be modified to include an aptamer.
  • the Type II endonuclease of the disclosure can be further fused to domains, e.g. catalytic domains to produce dual action Cas proteins.
  • a Type II endonuclease is further fused to a base editor.
  • gRNAs DNA-targeting RNAs that direct the activities of the novel Type II endonucleases of the disclosure to a specific target sequence within a target DNA.
  • DNA- targeting RNAs are referred to herein as “gRNAs” or “gRNAs”
  • gRNAs DNA-targeting RNAs
  • a Type II gRNA comprises a first segment (also referred to herein as a “targeter-RNA”, a "DNA-targeting segment” or a "DNA-targeting sequence”) and a second segment (also referred to herein as a “activator- RNA”, a "activator-RNA” or a "protein-binding sequence”).
  • nucleotide sequences encoding the Type II gRNAs of the disclosure. i. Targeter-RNA
  • the targeter-RNA of a Type II endonulcease gRNA of the disclosure comprises a nucleotide sequence that is complementary to a sequence in a target DNA (targeting sequence of the gRNA; DNA- targeting sequence; spacer sequence).
  • the targeter-RNA can interchangeably be referred to as a crRNA.
  • the targeter-RNA of a gRNA interacts with a target DNA in a sequence-specific manner via hybridization (i.e., base pairing).
  • the nucleotide sequence of the targeter-RNA may vary and determines the location within the target DNA that the gRNA and the target DNA will interact.
  • the targeter-RNA of a subject gRNA can be modified (e.g., by genetic engineering) to hybridize to any desired sequence within a target DNA.
  • a naturally unprocessed pre-crRNA of Type II comprises a direct repeat and an adjacent spacer (the portion of the crRNA that allows for targeting to a DNA molecule).
  • direct repeats partial sequence or entire sequence
  • Exemplary direct repeat sequences include SEQ ID NO: 115, 120, 125, and 130. It is noted that while the exemplary sequences are provided in DNA nucleotides, it is understood that this DNA can then be transcribed into RNA.
  • the mature guides of disclosure may incorporate the entire or partial sequence of the exemplary direct repeat sequences provided herein; the guides may be composed of DNA nucleotides, analogous RNA nucleotides, or a combination of DNA and RNA nucleotides.
  • Exemplary predicted secondary structures of the pre-crRNAs of the Type II endonucleases of the disclosure are presented in FIGS. 55, 58, 61, and 64.
  • the targeter-RNA can have a length of from about 12 nucleotides to about 100 nucleotides.
  • the targeter-RNA can have a length of from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50nt, from about 12 nt to about 40 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, or from about 12 nt to about 19 nt.
  • the targeter-RNA can have a length of from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 19 nt to about 70 nt, from about 19 nt to about 80 nt, from about 19 nt to about 90 nt, from about 19 nt to about 100 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, from about 20 nt to
  • the gRNAs of the disclosure include a portion of, or the entirety of the naturally occurring direct repeat sequences which can be incorporated into the engineered gRNAs of the disclosure.
  • Exemplary Type II naturally occurring direct sequences are provided herein, and include SEQ ID NO: and 115, 120, 125, and 130.
  • FIGS. 55, 58, 61, and 64 provide exemplary predicted secondary structures of the direct repeats of the disclosure.
  • the gRNAs of the disclosure include non-naturally occurring, engineered direct repeat sequences which can be incorporated into the engineered gRNAs of the disclosure. ii. Spacer Sequences
  • gRNAs of the disclosure comprise spacer sequences, complementary to the target DNA. More specifically, the nucleotide sequence of the targeter-RNA that is complementary to a target nucleotide sequence (the DNA-targeting sequence or spacer sequence) of the target DNA can have a length at least about 12 nt.
  • the DNA-targeting sequence of the targeter-RNA that is complementary to a target sequence of the target DNA can have a length at least about 12 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt or at least about 40 nt.
  • the DNA-targeting sequence of the targeter-RNA that is complementary to a target sequence of the target DNA can have a length of from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 5 Ont, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60
  • the nucleotide sequence (the DNA-targeting sequence) of the targeter-RNA that is complementary to a nucleotide sequence (target sequence) of the target DNA can have a length at least about 12 nt. In some embodiments, the DNA-targeting sequence of the targeter-RNA that is complementary to a target sequence of the target DNA is 20 nucleotides in length. In some embodiments, the DNA-targeting sequence of the targeter-RNA that is complementary to a target sequence of the target DNA is 19 nucleotides in length.
  • the percent complementarity between the spacer sequence of the targeter-RNA and the target sequence of the target DNA can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).
  • the percent complementarity between the DNA-targeting sequence of the targeter- RNA and the target sequence of the target DNA is 100% over the 1-25 contiguous 5 '-most nucleotides of the target sequence of the complementary strand of the target DNA.
  • the percent complementarity between the DNA-targeting sequence of the targeter-RNA and the target sequence of the target DNA is at least 60% over about 1 - 25 contiguous nucleotides. In some embodiments, the percent complementarity between the DNA-targeting sequence of the targeter-RNA and the target sequence of the target DNA is 100% over the 1 - 25 contiguous 5 '-most nucleotides of the target sequence of the complementary strand of the target DNA and as low as 0% over the remainder. In such a case, the DNA-targeting sequence can be considered to be 1 - 25 nucleotides in length.
  • the spacer sequence of a Type II gRNA of the disclosure is directed to a target sequence in a mammalian organism. In some embodiments the spacer sequence is directed to a target sequence in a non-mammalian organism. [0294] In some embodiments, the spacer sequence of a Type II gRNA of the disclosure is directed to a target sequence which is a sequence of a human. In some embodiments, the target sequence is a sequence of a non -human primate.
  • the spacer sequence of a Type II gRNA of the disclosure is directed to a target sequence selected of a therapeutic target.
  • the spacer sequence of a Type II gRNA of the disclosure is directed to a target sequence selected of a diagnostic target - for example in such embodiments a labeled catalytically dead Type II endonuclease of the disclosure and a gRNA directed to a diagnostic target DNA is contacted with the target DNA, or a cell comprising the target DNA, or a sample comprising the target DNA.
  • the activator-RNA of a Type II gRNA of the disclosure binds with its cognate Type II endonuclease of the disclosure.
  • the activator-RNA can interchangeably be referred to as a tracrRNA.
  • the gRNA guides the bound Type II endonuclease to a specific nucleotide sequence within target DNA via the above described targeter-RNA.
  • the activator-RNA of a Type II gRNA comprises two stretches of nucleotides that are complementary to one another.
  • Exemplary tracrRNAs are provided herein, and include SEQ ID NO: 114, 119, 124, and 129.
  • FIGS. 55, 58, 61, and 64 provide exemplary predicted secondary structures of the tracrRNAs of the disclosure. iv. Dual-molecule Type II gRNAs
  • dual molecule (two-molecule) gRNAs for the novel Type II endonucleases of the disclosure.
  • Such gRNAs comprise two separate RNA molecules (activator RNA-tracRNA; and the targeting RNA- crRNA).
  • Each of the two RNA molecules of a subject doublemolecule gRNA comprises a stretch of nucleotides that are complementary to one another such that the complementary nucleotides of the two RNA molecules hybridize to form the double stranded RNA duplex of the gRNA.
  • a dual-molecule gRNA can be designed to allow for controlled (i.e., conditional) binding of a targeter-RNA with an activator-RNA. Because a dual-molecule gRNA is not functional unless both the activator-RNA and the targeter-RNA are bound in a functional complex with Type II endonulcease of the disclosure, a dual-molecule gRNA can be inducible (e.g., drug inducible) by rendering the binding between the activator-RNA and the targeter-RNA to be inducible. As one non-limiting example, RNA aptamers can be used to regulate (i.e., control) the binding of the activator-RNA with the targeter-RNA. Accordingly, the activator-RNA and/or the targeter-RNA can comprise an RNA aptamer sequence. [0300] The dual-molecule guide can be modified to include an aptamer v. Single-molecule Type II endonulcease gRNAs
  • Type II gRNAs that comprises a single-molecule gRNA (interchangeably referred to herein as a sgRNA), for the novel Type II endonucleases of the disclosure.
  • an engineered single-molecule gRNA comprising: a. a targeter-RNA that is capable of hybridizing with a target sequence in a target DNA; and b. an activator-RNA that is capable of hybridizing with the targeter-RNA to form a doublestranded RNA duplex, the activator-RNA comprising a activator-RNA, wherein the targeter-RNA and the activator-RNA are covalently linked to one another, wherein the single-molecule gRNA is capable of forming a complex with a novel Type II endonuclease of the disclosure, and wherein hybridization of the targeter-RNA to the target sequence is capable of targeting the Type II endonuclease of the disclosure to the target DNA.
  • a subject single-molecule gRNA comprises two segments of nucleotides (a targeter-RNA and an activator-RNA) that are complementary to one another, can be covalently linked by intervening nucleotides ("linkers” or “linker nucleotides”), and hybridize to form the double stranded RNA duplex (dsRNA duplex) of the activator-RNA, whereby resulting in a stem-loop structure.
  • the targeter-RNA and the activator-RNA are covalently linked via the 3' end of the targeter-RNA and the 5' end of the activator-RNA.
  • the activator-RNA is covalently linked via the 5' end of the targeter-RNA and the 3' end of the activator-RNA.
  • the targeter-RNA and the activator-RNA are arranged in a 5’ to 3’ orientation.
  • the activator-RNA and the targeter-RNA are arranged in a 5’ to 3’ orientation.
  • the single molecule gRNA comprises one or more sequence modifications compared to a sequence of a corresponding wild type tracrRNA and/or crRNA.
  • the targeter-RNA and the activator-RNA are covalently linked to one another via a linker.
  • the linker of a single-molecule gRNA can have a length of from about 3 nucleotides to about 30 nucleotides. In exemplary embodiments, the linker of a single- molecule gRNA is 4, 5, 6, or 7 nt.
  • An exemplary single-molecule gRNA comprises two complementary stretches of nucleotides that hybridize to form a dsRNA duplex. In some embodiments, one of the two complementary stretches of nucleotides of the single -molecule gRNA (or the DNA encoding the stretch) is at least about 60% identical to one of the activator-RNA.
  • one of the two complementary stretches of nucleotides of the single-molecule gRNA is at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical or 100 % identical to an activator-RNA.
  • the activator-RNA and targeter-RNA segments can be engineered, while ensuring that the structure of the protein-binding domain of the gRNA is conserved.
  • RNA folding structure of a naturally occurring protein-binding domain of a DNA-targeting RNA can be taken into account in order to design artificial protein- binding domains (either dual-molecule or single-molecule versions).
  • the activator-RNA in a single-molecule gRNA can have a length of from about 10 nucleotides to about 100 nucleotides.
  • the activator-RNA can have a length of from about 15 nucleotides (nt) to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt or from about 15 nt to about 25 nt.
  • the dsRNA duplex of the activator-RNA can have a length from about 6 nucleotides (nt) to about 50bp.
  • the dsRNA duplex of the activator-RNA can have a length from about 6 nt to about 40 nt, from about 6 nt to about 30bp, from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8 nt to about 30bp, from about 8 nt to about 25 nt, from about 8 nt to about 20 nt or from about 8 nt to about 15 nt.
  • the dsRNA duplex of the activator-RNA can have a length from about from about 8 nt to about 10 nt, from about 10 nt to about 15 nt, from about 15 nt to about 18 nt, from about 18 nt to about 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt, from about 35 nt to about 40 nt, or from about 40 nt to about 50 nt.
  • the dsRNA duplex of the activator- RNA has a length of 8 - 15 base pairs.
  • the percent complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the activator-RNA can be at least about 60%.
  • the percent complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the activator-RNA can be at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% .
  • the percent complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the activator-RNA is 100%.
  • the spacer sequence of a Type II gRNA (whether it is a single molecule gRNA or a dual molecule gRNA) of the disclosure is directed to a target sequence in a mammalian organism, e.g. a human or non-human primate. In some embodiments, the spacer sequence of a Type II gRNA of the disclosure is directed to a target sequence in a bacteria.
  • the spacer sequence of a Type II gRNA of the disclosure is directed to a target sequence in a virus. In some embodiments, the spacer sequence of a Type II gRNA of the disclosure is directed to a target sequence in a plant.
  • the single-molecule Type II gRNAs of the disclosure can be modified to include an aptamer.
  • Type II gRNAs of the disclosure can be provided as gRNA arrays.
  • gRNA arrays include more than one gRNA arrayed in tandem, and can be processed into into two or more individual gRNAs.
  • a precursor Type II gRNA array comprises two or more (e.g., 3 or more, 4 or more, 5 or more, 2, 3, 4, or 5) gRNAs (e.g., arrayed in tandem as precursor molecules).
  • two or more gRNAs can be present on an array (a precursor gRNA array).
  • a Type II endonuclease of the disclosure can cleave the precursor gRNA array into individual gRNAs.
  • a gRNA array includes 2 or more gRNAs (e.g., 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, gRNAs).
  • the gRNAs of a given array can target (i.e., can include guide sequences that hybridize to) different target sites of the same target DNA.
  • two or more gRNAs of a precursor gRNA array have the same guide sequence.
  • the precursor gRNA array comprises two or more gRNAs that target different target sites within the same target DNA.
  • the precursor gRNA array comprises two or more gRNAs that target different target DNAs.
  • the target DNA is part of a chromosome in vitro. In some embodiments, the target DNA is part of a chromosome in vivo.
  • the target DNA is part of a chromosome in a cell.
  • the target DNA is extrachromosomal DNA.
  • the target DNA is in a cell, wherein the cell is selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, a mammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non -human primate cell, and a human cell.
  • the cell is selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an
  • the target DNA is the DNA of a parasite.
  • the target DNA is a viral DNA.
  • the target DNA is a bacterial DNA.
  • the modifying comprises introducing a double strand break in the target
  • the contacting occurs under conditions that are permissive for nonhom ologous end joining or homology-directed repair.
  • the method comprises contacting the target DNA with a donor polynucleotide, wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA.
  • the method does not comprise contacting the cell with a donor polynucleotide, wherein the target DNA is modified such that nucleotides within the target DNA are deleted.
  • the novel Type VI endonucleases of the disclosure for the modification of a target RNA.
  • the method of modifying a target RNA comprising contacting the target RNA with any one of the Type VI systems described herein.
  • the target RNA is in vitro. In some embodiments, the target RNA in vivo.
  • the target RNA is in a cell, wherein the cell is selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, a mammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non -human primate cell, and a human cell.
  • the cell is selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell,
  • the target RNA is the RNA of a parasite.
  • the target RNA is a viral RNA.
  • the target RNA is a bacterial RNA.
  • the target RNA may be any suitable form of RNA. This may include, in some embodiments, mRNA. In other embodiments, the target RNA may include tRNA or rRNA. In other embodiments, the target RNA may include miRNA. In other embodiments, the target RNA may include siRNA. c. Therapeutic Applications (Type II, Type V endonucleases)
  • the disclosure provides novel Type II, and Type V endonucleases, engineered systems, one or more polynucleotides encoding components of said system, and vector or delivery systems comprising one or more polynucleotides encoding components of said system for use in therapeutic methods.
  • the therapeutic methods may comprise gene or genome editing, or gene therapy.
  • the therapeutic methods comprise use and delivery of the novel Type II or Type V endonucleases of the disclosure.
  • a method of modifying a target DNA comprising contacting a target DNA, a cell comprising the target DNA, or a subject with cells with the target DNA, with any one of the Type II and Type V systems described herein.
  • a method of modifying a target RNA the method comprising contacting a target RNA, a cell comprising the target RNA, or a subject with cells with the target RNA, with any one of the Type VI systems described herein.
  • the target DNA is part of a chromosome in vitro. In some embodiments, the target DNA is part of a chromosome in vivo.
  • the target DNA is part of a chromosome in a cell.
  • the target DNA is extrachromosomal DNA.
  • the target DNA is in a cell, wherein the cell is selected from the group consisting of an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, a mammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non -human primate cell, and a human cell.
  • the cell is selected from the group consisting of an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell
  • the target DNA is outside of a cell.
  • the target DNA is in vitro inside of a cell.
  • the target DNA is in vivo, inside of a cell.
  • the modifying comprises introducing a double strand break in the target DNA.
  • the contacting occurs under conditions that are permissive for nonhom ologous end joining or homology-directed repair.
  • the method comprises contacting the target DNA with a donor polynucleotide, wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA.
  • the method does not comprise contacting the cell with a donor polynucleotide, wherein the target DNA is modified such that nucleotides within the target DNA are deleted.
  • the therapeutic methods involve modifying a target DNA comprising a target sequence of a gene of interest and/or the regulatory region of the gene of interest, the method comprising delivering to a cell comprising the target DNA, a Type II endonuclease of the disclosure and one or more Type II gRNAs, a Type V endonuclease of the disclosure and one or more Type V gRNAs, one or more nucleotides encoding the Type II endonuclease of the disclosure and one or more Type II gRNAs, or one or more nucleotides encoding a Type V endonuclease of the disclosure and one or more Type V gRNAs.
  • the gene of interest is within a eukaryotic cell, e.g. a human or nonhuman primate cell.
  • the gene of interest is within a plant cell.
  • the delivering comprises delivering to the cell a Type II endonuclease of the disclosure (or one or more nucleotides encoding the same) and one or more Type II gRNAs.
  • the delivering comprises delivering to the cell a Type V endonuclease of the disclosure (or one or more nucleotides encoding the same) and one or more Type V gRNAs.
  • the delivering comprises delivering to the cell one or more nucleotides encoding the Type II endonuclease of the disclosure and one or more Type II gRNAs.
  • the delivering comprises delivering to the cell one or more nucleotides encoding a Type V endonuclease of the disclosure and one or more Type V gRNAs.
  • the disclosure provides novel Type VI endonucleases, engineered systems, one or more polynucleotides encoding components of said system, and vector or delivery systems comprising one or more polynucleotides encoding components of said system for use in therapeutic methods.
  • a method of modifying a target RNA comprising contacting a target RNA, a cell comprising the target RNA, or a subject with cells with the target RNA, with any one of the Type VI systems described herein.
  • a method of modifying a target RNA the method comprising contacting a target RNA, a cell comprising the target RNA, or a subject with cells with the target RNA, with any one of the Type VI systems described herein.
  • the target RNA is in a cell, wherein the cell is selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, a mammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non -human primate cell, and a human cell.
  • the cell is selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell,
  • the target RNA is outside of a cell.
  • the target RNA is in vitro inside of a cell.
  • the target RNA is in vivo, inside of a cell.
  • the target RNA may be any suitable form of RNA. This may include, in some embodiments, mRNA. In other embodiments, the target RNA may include tRNA or rRNA. In other embodiments, the target RNA may include miRNA. In other embodiments, the target RNA may include siRNA.
  • the therapeutic methods involve modifying a target RNA comprising a mRNA encoding a gene of interest and/or the regulatory region of the mRNA of interest, the method comprising delivering to a cell comprising the target RNA, a Type VI endonuclease of the disclosure and one or more Type VI gRNAs, or one or more nucleotides encoding the Type VI endonuclease of the disclosure and one or more Type VI gRNAs.
  • the RNA of interest is within a eukaryotic cell, e.g. a human or nonhuman primate cell.
  • the RNA of interest is within a plant cell.
  • the delivering comprises delivering to the cell a Type VI endonuclease of the disclosure (or one or more nucleotides encoding the same) and one or more Type VI gRNAs. [0369] In some embodiments, the delivering comprises delivering to the cell one or more nucleotides encoding a Type VI endonuclease of the disclosure and one or more Type VI gRNAs. e. Delivery
  • Delivery of the Type II, Type V, and Type VI components to a cell can be achieved by any variety of delivery methods known to those of skill in the art.
  • the components can be combined with a lipid.
  • the components combined with a particle, or formulated into a particle, e.g. a nanoparticle.
  • Methods of introducing a nucleic acid and/or protein into a host cell are known in the art, and any convenient method can be used to introduce a subject nucleic acid (e.g., an expression construct/vector) into a target cell (e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, mammalian cell, human cell, and the like).
  • a subject nucleic acid e.g., an expression construct/vector
  • a target cell e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, mammalian cell, human cell, and the like.
  • Suitable methods include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome- mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery and the like.
  • PEI polyethyleneimine
  • a gRNA can be introduced, e.g., as a DNA molecule encoding the gRNA, or can be provided directly as an RNA molecule (or a chimeric/hybrid molecule when applicable).
  • Type II, Type V, or Type VI endonuclease is provided as a nucleic acid (e.g., an mRNA, a DNA, a plasmid, an expression vector, a viral vector, etc.) that encodes the protein.
  • the Type II, Type V, or Type VI endonuclease is provided directly as a protein (e.g., without an associated gRNA or with an associate gRNA, i.e., as a ribonucleoprotein complex — RNP).
  • a Type II, Type V, or Type VI endonuclease of the disclosure can be introduced into a cell (provided to the cell) by any convenient method; such methods are known to those of ordinary skill in the art.
  • a Type II, Type V, or Type VI endonuclease of the disclosure can be injected directly into a cell (e.g., with or without a gRNA or nucleic acid encoding a gRNA).
  • a pre-formed complex of a Type II, Type V, or Type VI endonuclease and a gRNA can be introduced into a cell (e.g., eukaryotic cell) (e.g., via injection, via nucleofection; via a protein transduction domain (PTD) conjugated to one or more components, e.g., conjugated to the Type II, Type V, or Type VI endonuclease of the disclosure, conjugated to a gRNA; etc.).
  • a cell e.g., eukaryotic cell
  • PTD protein transduction domain
  • a nucleic acid e.g., a gRNA; a nucleic acid comprising a nucleotide sequence encoding a Type II, Type V, or Type VI endonuclease of the disclosure; etc.
  • a polypeptide e.g., a Type II, Type V, or Type VI endonuclease of the disclosure
  • a cell e.g., a target host cell
  • the particle is a nanoparticle.
  • a Type II, Type V, or Type VI endonuclease of the disclosure may be delivered simultaneously using particles or lipid envelopes.
  • gRNA or a nucleic acid such as one or more expression vectors encoding the gRNA
  • Suitable target cells include, but are not limited to: a bacterial cell; an archaeal cell; a cell of a single-cell eukaryotic organism; a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell (e.g., a yeast cell); an animal cell; a cell from an invertebrate animal (e.g.
  • a cell of an insect e.g., a mosquito; a bee; an agricultural pest; etc.
  • a cell of an arachnid e.g., a spider; a tick; etc.
  • a cell from a vertebrate animal e.g., a fish, an amphibian, a reptile, a bird, a mammal
  • a cell from a mammal e.g., a cell from a rodent; a cell from a human; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse, a rat); a cell of a lagomorph (e.g., a rabbit); a cell of an ungulate (e.g., a cow, a horse, a camel, a llama, a vicuna,
  • a stem cell e.g. an embryonic stem (ES) cell, an induced pluripotent stem cell (iPSC), a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somatic cell, e.g.
  • ES embryonic stem
  • iPSC induced pluripotent stem cell
  • germ cell e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.
  • an adult stem cell e.g.
  • a fibroblast a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.).
  • Cells may be from cell lines or primary cells.
  • Target cells can be unicellular organisms and/or can be grown in culture. If the cells are primary cells, they may be harvest from an individual by any convenient method. For example, leukocytes may be conveniently harvested by apheresis, leukocytapheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be conveniently harvested by biopsy.
  • a mitotic and/or post-mitotic cell of interest in the disclosed methods may include a cell of any organism (e.g.
  • a bacterial cell e.g., a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like, a fungal cell (e.g., a yeast cell), an animal cell, a cell of an invertebrate animal (e.g.
  • fruit fly cnidarian, echinoderm, nematode, etc.
  • a cell of a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell of a mammal a cell of a rodent, a cell of a human, etc.
  • Plant cells include cells of a monocotyledon, and cells of a dicotyledon.
  • the cells can be root cells, leaf cells, cells of the xylem, cells of the phloem, cells of the cambium, apical meristem cells, parenchyma cells, collenchyma cells, sclerenchyma cells, and the like.
  • Plant cells include cells of agricultural crops such as wheat, com, rice, sorghum, millet, soybean, etc.
  • Plant cells include cells of agricultural fruit and nut plants, e.g., plant that produce apricots, oranges, lemons, apples, plums, pears, almonds, etc.
  • Non-limiting examples of cells include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatos, rice, cassava, sugarcane, pumpkin, hay, potatos, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, homworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C.
  • seaweeds e.g. kelp
  • a fungal cell e.g., a yeast cell, a cell from a mushroom
  • an animal cell e.g., a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.)
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like.
  • the cell is a cell that does not originate from a natural organism (e.g.,
  • a cell can be an in vitro cell (e.g., established cultured cell line).
  • a cell can be an ex vivo cell (cultured cell from an individual).
  • a cell can be and in vivo cell (e.g., a cell in an individual).
  • a cell can be an isolated cell.
  • a cell can be a cell inside of an organism.
  • a cell can be an organism.
  • Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autotransplated expanded cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and
  • the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell.
  • the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage.
  • the immune cell is a cytotoxic T cell.
  • the immune cell is a helper T cell.
  • the immune cell is a regulatory T cell (Treg).
  • the cell is a stem cell.
  • Stem cells include adult stem cells.
  • Adult stem cells are also referred to as somatic stem cells.
  • Adult stem cells are resident in differentiated tissue, but retain the properties of self-renewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found.
  • somatic stem cells include muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.
  • Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc.
  • the stem cell is a human stem cell.
  • the stem cell is a rodent (e.g., a mouse; a rat) stem cell.
  • the stem cell is a non-human primate stem cell.
  • Any gene of interest can serve as a target for modification.
  • the target is a gene or mRNA implicated in cancer.
  • the target is a gene or mRNA implicated in an immune disease, e.g. an autoimmune disease.
  • the target is a gene or mRNA implicated in a neurodegenerative disease.
  • the target is a gene or mRNA implicated in a neuropsychiatric disease.
  • the target is a gene or mRNA implicated in a muscular disease.
  • the target is a gene or mRNA implicated in a cardiac disease.
  • the target is a gene implicated in diabetes.
  • the target is a gene implicated in kidney disease.
  • the therapeutic methods provided herein can include delivery of precursor gRNA arrays.
  • a Type II, Type V, or Type VI endonuclease of the disclosure can cleave a precursor gRNA into a mature gRNA, e.g., by endorib onucleoly tic cleavage of the precursor.
  • a Type II, Type V, or Type VI endonuclease of the disclosure can cleave a precursor gRNA array (that includes more than one gRNA arrayed in tandem) into two or more individual gRNAs.
  • Type V or Type VI endonucleases of the disclosure also possess collateral (trans-cleavage activity), i.e. the ability to promiscuously cleave non-targeted oligonucleotides, once activated by detection of a target DNA or RNA.
  • collateral trans-cleavage activity
  • Type V or Type VI endonuclease of the disclosure is activated by a gRNA, which occurs when a sample includes a target sequence to which the gRNA hybridizes (i.e., the sample includes the targeted DNA or the targeted RNA), the Type V or Type VI becomes a nuclease that promiscuously cleaves single stranded oligonucleotides (i.e., non-target single stranded oligonucleotides, i.e., single stranded oligonucleotides to which the guide sequence of the gRNA does not hybridize).
  • the result can be cleavage (collateral) of oligonucleotidesin the sample, which can be detected using any convenient detection method (e.g., using a labeled single stranded detector DNA, labeled detector RNA, or labeled detector DNA/RNA chimeric oligonucleotides).
  • RNA in a sample.
  • methods and compositions for detecting a target DNA (dsDNA or ssDNA) or RNA in a sample are also provided.
  • methods and compositions for cleaving non-target oligonucleotides are also provided.
  • a “detector” comprises an oligonucleotide of any nature, single or double stranded and does not hybridize with the guide sequence of the gRNA (i.e., the detector oligonucleotide that is a non-target).
  • the detection methods based on the collateral activity of the Type V or Type VI endonucleases of the disclosure can include:
  • Type V or Type VI endonuclease is activated by a gRNA, which can occur when the sample includes a target DNA to which the gRNA hybridizes (i.e., the sample includes the targeted sequence in the target DNA)
  • the Type V or Type VI can be activated to function as an endoribonuclease that non-specifically cleaves detector oligonucleotides (including non-target ss oligonucleotides) present in the sample.
  • the result is cleavage of a detector oligonucleotide in the sample, which can be detected using any convenient detection method (e.g., using a labeled detector oligonucleotides).
  • detector oligonucleotides e.g., ssDNAs, ssRNAs, ssDNA/RNA chimeras or detectors comprising ss and ds regions.
  • Such methods can include contacting a population of nucleic acids, wherein said population comprises a target DNA and a plurality of non-target ss oligonucleotides, with: (i) a Type V or Type VI endonuclease of the disclosure; and (ii) a gRNA comprising: a region that binds to the Type V or Type VI effector protein, and a guide sequence that hybridizes with the target DNA, wherein the Type V or Type VI endonuclease cleaves non-target ss oligonucleotides
  • a method of detecting a target DNA or RNA in a sample comprising:
  • a gRNA comprising a spacer sequence that is capable of hybridizing with a target sequence in a target DNA or RNA
  • the contacting step can be carried out in an acellular environment, e.g., outside of a cell. In other embodiments, 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 in vivo.
  • the contacting step of a detection method can be carried out in a composition comprising divalent metal ions.
  • the gRNA can be provided as RNA or as a nucleic acid encoding the gRNA (e.g., a DNA such as a recombinant expression vector), described herein.
  • the contacting, prior to the measuring step can last for any period of time, e.g from 5 seconds to 2 hours or more, prior to the measuring step.
  • the sample is contacted for 45 minutes or less prior to the measuring step.
  • the sample is contacted for 30 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.
  • the sample is contacted for 1 minute or less prior to the measuring step.
  • the sample is contacted for from 50 seconds to 60 seconds prior to the measuring step.
  • the sample is contacted for from 40 seconds to 50 seconds prior to the measuring step.
  • the sample is contacted for from 30 seconds to 40 seconds prior to the measuring step. In some embodiments the sample is contacted for from 20 seconds to 30 seconds prior to the measuring step. In some embodiments the sample is contacted for from 10 seconds to 20 seconds prior to the measuring step.
  • the detection methods provided herein can detect a target DNA or RNA with a high degree of sensitivity. Accordingly, in some embodiments, the detection methods of the disclosure can be used to detect a target DNA or RNA present in a sample comprising a plurality of DNA or RNA (including the target DNA or RNA and a plurality of non-target DNAs or RNAs), where the target DNA or RNA is present at one or more copies per 5 to 10 A 9 copies of the non-target DNAs or RNAs).
  • the threshold of detection for a detection method of detecting a target DNA or RNA in a sample, is 10 nM or less.
  • the term “threshold of detection” is used herein to describe the minimal amount of target DNA or RNA that must be present in a sample in order for detection to occur.
  • a subject composition or method exhibits an attomolar (aM) sensitivity of detection.
  • a subject composition or method exhibits a femtomolar (fM) sensitivity of detection.
  • a subject composition or method exhibits a picomolar (pM) sensitivity of detection.
  • a subject composition or method exhibits a nanomolar (nM) sensitivity of detection.
  • a target DNA can be single stranded (ssDNA) or double stranded (dsDNA). There need not be any preference or requirement for a PAM sequence in a single stranded target DNA.
  • a target RNA can be single stranded RNA.
  • the source of the target DNA or RNA can be any source.
  • the target DNA or RNA is a viral or bacterial DNA or RNA (e.g., a genomic DNA or RNA of a DNA or RNA virus or bacteria).
  • detection method can be for detecting the presence of a viral or bacterial DNA amongst a population of nucleic acids (e.g., in a sample).
  • a RNA-carrying organism for example, a RNA virus (e.g. a coronavirus) - it is understood that a step such as reverse transcription may be carried out on a sample comprising the RNA-carrying organism to generated cDNA, and the cDNA is then the target DNA.
  • the RNA can also be detected directly using a Type VI endonuclease of the disclosure.
  • Exemplary non-limiting sources for target DNA or RNA are provided in Tables 10a - lOf.
  • an in vitro transcription (IVT) step could be included to transcribe the genome to RNA, prior to assessment.
  • IVT in vitro transcription
  • a reverse transcriptase (RT) step could be included to reverse transcribe the genome to DNA, prior to assessment.
  • DNA or RNA obtained from viruses and bacteria related to respiratory infections may also be targeted.
  • a list of targets of interest may include the examples shown in Table 10c.
  • DNA or RNA obtained from viruses and bacteria related to sexually transmitted diseases may also be targeted.
  • a list of targets of interest may include the examples shown in Table lOd.
  • DNA or RNA targets may also be targeted.
  • male genes to determine the sex of the embryo of a pregnant woman/animal, and the male genes to determine the sex of plants and seeds may also be targeted.
  • further targets of interest may include the following shown in Table lOe. Table lOe
  • sample is used herein to mean any sample that includes DNA or RNA (e.g., in order to determine whether a target DNA or RNA is present among a population of DNA or RNAs).
  • the DNA can be single stranded, double stranded DNA, complementary DNA, and the like.
  • a sample intended for detection comprises a plurality of nucleic acids.
  • 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., DNA or RNAs).
  • a detection method can be used as a very sensitive way to detect a target DNA or RNA present in a sample (e.g., in a complex mixture of nucleic acids such as DNA or RNAs).
  • the sample includes 5 or more DNA or RNAs (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 DNA or 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 A 3 or more, 5* 10 A 3 or more, 10 A 4 or more, 5* 10 A 4 or more, 10 A 5 or more, 5* 10 A 5 or more, 10 A 6 or more 5* 10 A 6 or more, or 10 A 7 or more, DNA or RNAs.
  • the sample comprises from 10 to 20, from 20 to 50, from 50 to 100, from 100 to 500, from 500 to 10 A 3, from 10 A 3 to 5* 10 A 3, from 5* 10 A 3 to 10 A 4, from 10 A 4 to 5* 10 A 4, from 5* 10 A 4 to 10 A 5, from 10 A 5 to 5* 10 A 5, from 5* 10 A 5 to 10 A 6, from 10 A 6 to 5* 10 A 6, or from 5* 10 A 6 to 10 A 7, or more than 10 A 7, DNA or RNAs.
  • the sample comprises from 5 to 10 A 7 DNA or RNAs (e.g., that differ from one another in sequence)(e.g., from 5 to 10 A 6, from 5 to 10 A 5, from 5 to 50,000, from 5 to 30,000, from 10 to 10 A 6, from 10 to 10 A 5, from 10 to 50,000, from 10 to 30,000, from 20 to 10 A 6, from 20 to 10 A 5, from 20 to 50,000, or from 20 to 30,000 DNA or RNAs).
  • 5 to 10 A 7 DNA or RNAs (e.g., that differ from one another in sequence)(e.g., from 5 to 10 A 6, from 5 to 10 A 5, from 5 to 50,000, from 5 to 30,000, from 10 to 10 A 6, from 10 to 10 A 5, from 10 to 50,000, from 10 to 30,000, from 20 to 10 A 6, from 20 to 10 A 5, from 20 to 50,000, or from 20 to 30,000 DNA or RNAs).
  • the sample includes 20 or more DNA or RNAs that differ from one another in sequence.
  • the sample includes DNA or RNAs 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, and the like.
  • the sample includes DNA or RNA from a cell such as a eukaryotic cell, e.g., a mammalian cell such as a human cell.
  • the sample can be derived from any source, e.g., the sample can be a synthetic combination of purified DNA or RNAs; the sample can be a cell lysate, a DNA or RNA-enriched cell lysate, or DNA or RNAs 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.
  • a sample can include a target DNA or RNA and a plurality of non-target DNA or RNAs. In some embodiments, the target DNA or RNA is present in the sample at one or more copies per 5 to 10 A 9 copies of the non-target DNA or RNAs.
  • Suitable samples include but are not limited to urine, blood, serum, plasma, lymphatic fluid, cerebrospinal fluid, saliva, nasopharyngeal, oropharyngeal, nasopharyngeal/ oropharyngeal, aspirate, or biopsy sample.
  • 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. Samples also can be 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.
  • samples can be obtained by use of a swab, for example, a nasopharyngeal swab, an oropharyngeal swab, or a nasopharyngeal/ oropharyngeal swab.
  • Samples also can be samples that have been enriched for particular types of molecules, e.g., DNA or RNAs. Samples 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 DNA or RNAs that is obtained from such cells (e.g., a cell lysate or other cell extract comprising DNA or RNAs).
  • 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 single-cell eukaryotic organisms; a plant or a plant cell; an algal cell; a fungal cell; an animal cell, tissue, or organ; a cell, tissue, or organ from an invertebrate animal; a cell, tissue, fluid, or organ from a vertebrate animal; a cell, tissue, fluid, or organ from a mammal (e.g., a human; a nonhuman 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.
  • 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.).
  • the source of the sample is a (or is suspected of being 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 (or is suspected of being a pathogen- infected cell, tissue, or organ.
  • the source of a sample can be an individual who may or may not be infected — and the sample could be any biological sample (e.g., blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage, sputum, a fecal sample, cerebrospinal fluid, a fine needle aspirate, a swab sample (e.g., a buccal swab, a cervical swab, a nasal swab), interstitial fluid, synovial fluid, nasal discharge, tears, buffy coat, a mucous membrane sample, an epithelial cell sample (e.g., epithelial cell scraping), etc.) collected from the individual.
  • the sample is a cell-free liquid sample.
  • the sample is a liquid sample that can comprise cells (urine, blood, serum, plasma, lymphatic fluid, cerebrospinal fluid, saliva, nasopharyngeal, oropharyngeal, nasopharyngeal/ oropharyngeal, aspirate, and biopsy).
  • 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 RNA or DNA viruses, e.g., coronoavirus (e.g.
  • SARS-CoV SARS-CoV-2, MERS-CoV
  • immunodeficiency virus e.g., HIV
  • influenza virus e.g., dengue; West Nile virus; herpes virus; yellow fever virus
  • Hepatitis Virus C Hepatitis Virus A
  • Hepatitis Virus B papillomavirus
  • Pathogenic viruses can include DNA viruses 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, pseudocowpox, bovine papular
  • Pathogens can include, e.g., DNAviruses [e.g.: 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, pseudo
  • the detection method generally includes a step of measuring (e.g., measuring a detectable signal produced by the Type V or Type VI of the disclosure.
  • a detectable signal can be any signal that is produced when ss oliogonucleotide is cleaved.
  • the step of detection can involve a fluorescence-based detection.
  • 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), the presence or absence of (or a particular amount of) a magnetic signal and the presence or absence of (or a particular amount of) an electrical signal.
  • a measured amount of detectable fluorescent signal e.g., 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), the presence or absence of (or a particular amount of) a magnetic signal and the presence or absence of (or a particular amount of) an electrical signal.
  • the measuring can in some embodiments be quantitative, e.g., in the sense that the amount of signal detected can be used to determine the amount of target DNA or RNA present in the sample.
  • the measuring can in some embodiments be qualitative, e.g., in the sense that the presence or absence of detectable signal can indicate the presence or absence of targeted DNA or RNA (e.g., virus, SNP, etc.).
  • a detectable signal will not be present (e.g., above a given threshold level) unless the targeted DNA or RNA(s) (e.g., virus, SNP, etc.) is present above a particular threshold concentration.
  • the threshold of detection can be titrated by modifying the amount of the Type V or Type VI endonuclease provided.
  • compositions and methods of this disclosure can be used to detect any DNA or RNA target.
  • the detection methods of the disclosure can be used to determine the amount of a target DNA or RNA in a sample (e.g., a sample comprising the target DNA or RNA and a plurality of non-target DNA or RNAs). Determining the amount of a target DNA or RNA 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 target DNA or RNA 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 target DNA or RNA present in the sample.
  • the detectable signal is detectable in less than 1, 2, 3, 4, 5, 10, 15, 20, 30, 60, 90, 120, 150, 180, 210, or 240 minutes.
  • sensitivity of a subject composition and/or method e.g., for detecting the presence of a target DNA or RNA, such as viral DNA or RNA or a SNP, in cellular genomic DNA or RNA
  • sensitivity of a subject composition and/or method can be increased by coupling detection with nucleic acid amplification.
  • the nucleic acids in a sample are amplified prior to contact with a Type V or Type VI; in particular embodiments, the Type V or Type VI remains in an inactive state until amplification has concluded.
  • the nucleic acids in a sample are amplified simultaneous with contact with Type V or Type VI.
  • Amplification can be carried out using primers. As it relates to the overall processing time for the detection method, amplification can occur for 5 seconds or more, up to 240 minutes or more.
  • Nucleic acid amplification can comprise polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), quantitative PCR (qPCR), reverse transcription qPCR (RT-qPCR), isothermal PCR, nested PCR, multiplex PCR, asymmetric PCR, touchdown PCR, random primer PCR, hemi-nested PCR, polymerase cycling assembly (PCA), colony PCR, ligase chain reaction (LCR), digital PCR, methylation specific-PCR (MSP),co-amplification at lower denaturation temperature-PCR (COLD-PCR), allele-specific PCR, intersequence-specific PCR (ISS-PCR), whole genome amplification (WGA), inverse PCR, and thermal asymmetric interlaced PCR (TAIL-PCR).
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription PCR
  • qPCR quantitative PCR
  • RT-qPCR reverse transcription qPCR
  • PCR reverse transcription qPCR
  • isothermal PCR nested PCR, multiple
  • the amplification is isothermal amplification.
  • Isothermal nucleic acid amplification methods can therefore be carried out inside or outside of a laboratory environment.
  • isothermal amplification methods include but are not limited to: loop-mediated isothermal Amplification (LAMP), helicase-dependent Amplification (HD A), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), Ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART), self-sustained sequence replication (3 SR), genome exponential amplification reaction (GEAR) and isothermal multiple displacement amplification (IMDA).
  • LAMP loop-mediated isothermal Amplification
  • a detection method includes contacting a sample with: i) a Type V or Type VI endonuclease of the disclosure; ii) a gRNA (or precursor gRNA array); and iii) a detector that does not hybridize with the guide sequence of the gRNA.
  • a detection method includes contacting a sample with a labeled detector that includes a fluorescenceemitting dye pair; the Type V or Type VI endonuclease of the disclosure has the ability to cleave the labeled detector after it is activated (by gRNA hybridizing to a target DNA or RNA); and the detectable signal that is measured is produced by the fluorescence-emitting dye pair.
  • a detection method includes contacting a sample with a labeled detector comprising a fluorescence resonance energy transfer (FRET) pair or a quencher/fluor pair, or both.
  • FRET fluorescence resonance energy transfer
  • a detection method includes contacting a sample with a labeled detector comprising a FRET pair. In some embodiments, a detection method includes contacting a sample with a labeled detector comprising a fluor/quencher pair.
  • Fluorescence-emitting dye pairs comprise a FRET pair or a quencher/fluor pair. In both embodiments 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.
  • the term “fluorescence-emitting dye pair” is a generic term used to encompass both a “fluorescence resonance energy transfer (FRET) pair” and a “quencher/fluor pair”.
  • FRET fluorescence resonance energy transfer
  • quencher/fluor pair The term “fluorescence-emitting dye pair” is used interchangeably with the phrase “a FRET pair and/or a quencher/fluor pair.”
  • the labeled detector 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 is cleaved.
  • the labeled detector produces a first detectable signal prior to being cleaved (e.g., from a FRET pair) and a second detectable signal when the labeled detector is cleaved (e.g., from a quencher/fluor pair).
  • the labeled detector comprises a FRET pair and a quencher/fluor pair.
  • the labeled detector comprises a FRET pair.
  • 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 11. FRET pairs provided in US10,253,365 are incorporate by reference herein in their entirety. In some embodiments, the FRET pair is 5' 6-FAM and 3IABkFQ (Iowa Black (Registred)-FQ). Table 11
  • FRET pairs Donor and and acceptor pairs
  • a detectable signal is produced when the labeled detector is cleaved (e.g., in some embodiments, the labeled detector comprises a quencher/fluor pair).
  • any fluorescent label can be utilized.
  • 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 Rhol l, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO 590, ATTO 594, ATTO Rhol3, ATTOTO 610, ATTO 620, ATTO Rhol4, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxal2, 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),
  • 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 540Q, ATTO 580Q, and ATTO 612Q
  • Dabsyl dimethylaminoazobenzenesulfonic acid
  • 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 540Q, ATTO 580Q, and ATTO 612Q
  • Dabsyl dimethylaminoazobenzenesulfonic acid
  • Iowa Black RQ Iowa Black FQ
  • cleavage of a labeled detector 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 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.
  • a labeled detector can be a nucleic acid mimetic.
  • Polynucleotide mimics include PNAs, LNAs, CeNAs, and morpholino nucleic acids.
  • a labeled detector can also include one or more substituted sugar moieties.
  • a labeled detector may also include modified nucleotides. e. Positive Controls
  • the detection methods provided herein can also include a positive control target DNA or RNA.
  • the methods include using a positive control gRNA that comprises a nucleotide sequence that hybridizes to a control target DNA or RNA.
  • the positive control target DNA or RNA is provided in various amounts.
  • the positive control target DNA or RNA is provided in various known concentrations, along with control non-target DNA or RNAs.
  • the method comprises contacting the sample with a precursor gRNA array, wherein the novel Type V or Type VI endonuclease of the disclosure cleaves the precursor gRNA array to produce said gRNA.
  • a such a gRNA array includes 2 or more gRNAs (e.g., 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, gRNAs).
  • the gRNAs of a given array can target (i.e., can include guide sequences that hybridize to) different target sites of the same target DNA or RNA (e.g., which can increase sensitivity of detection) and/or can target different target DNA or RNAs (e.g., single nucleotide polymorphisms (SNPs), different strains of a particular virus, etc.), and such could be used for example to detect multiple strains of a virus.
  • each gRNA of a precursor gRNA array has a different guide sequence.
  • the precursor gRNA array comprises two or more gRNAs that target different target sites within the same target DNA or RNA.
  • such a scenario can in some embodiments increase sensitivity of detection by activating Type II, Type V or Type VI endonuclease of the disclosure when either one hybridizes to the target DNA or RNA.
  • subject composition e.g., kit
  • method includes two or more gRNAs (in the context of a precursor gRNA array, or not in the context of a precursor gRNA array, e.g., the gRNAs can be mature gRNAs).
  • the precursor gRNA array comprises two or more gRNAs that target different target DNA or RNAs.
  • a scenario can result in a positive signal when any one of a family of potential target DNA or RNAs is present.
  • Such an array could be used for targeting a family of transcripts, e.g., based on variation such as single nucleotide polymorphisms (SNPs) (e.g., for diagnostic purposes). Such could also be useful for detecting whether any one of a number of different strains of virus is present.
  • SNPs single nucleotide polymorphisms
  • subject composition e.g., kit
  • method includes two or more gRNAs (in the context of a precursor gRNA array, or not in the context of a precursor gRNA array, e.g., the gRNAs can be mature gRNAs).
  • compositions and pharmaceutical compositions comprising the Type II, Type V, or Type VI endonucleases and/or the Type II, Type V, or Type VI gRNAs of the disclosure, which can optionally include a pharmaceutically acceptable carrier and/or a protein stabilizing buffer, and/or a nucleic acid stabilizing buffer.
  • the Type II, Type V, or Type VI endonucleases and/or the Type II, Type V, or Type VI gRNAs are provided in a lyophilized form.
  • compositions comprising gRNAs and/or gRNA arrays of the disclosure (compatible for use with Type II, Type V, or Type VI endonucleases of the disclosure), and optionally a protein stabilizing buffer.
  • proteins comprising an amino acid sequence with 30%-99.5% homology to any one of SEQ ID NOs: 1-20.
  • compositions comprising these proteins, and optionally a pharmaceutically acceptable carrier.
  • these proteins and optionally a protein stabilizing buffer.
  • DNA polynucleotides encoding a sequence that encodes any of the Type II, Type V, or Type VI endonucleases of the disclosure.
  • recombinant expression vectors comprising such DNA polynucleotides.
  • a nucleotide sequence encoding a Type II, Type V, or Type VI endonuclease of the disclosure is operably linked to a promoter.
  • the nucleic acid encoding the Type II, Type V, or Type VI endonuclease further comprises a nuclear localization signal (NLS), useful for expression in eukaryotic systems.
  • NLS nuclear localization signal
  • DNA polynucleotides or RNAs comprising a sequence that encodes any of the gRNAs of the disclosure. Also provided are recombinant expression vectors comprising such DNA polynucleotides. In some embodiments, a nucleotide sequence encoding a gRNA of the disclosure is operably linked to a promoter.
  • host cells comprising any of the recombinant vectors provided herein.
  • kits comprising one or more components of the Type II, Type V, and Type VI engineered systems described herein, useful for a variety of applications including, but not limited to, therapeutic and diagnostic applications.
  • kits comprising: (a) Type II endonuclease of the dislcosure, or a nucleic acid encoding the Type II endonuclease; and (b) Type II gRNA, wherein the gRNA and the Type II endonuclease do not naturally occur together, wherein the gRNA is capable of hybridizing to a target sequence in a target DNA, and the gRNA is capable of forming a complex with the Type II endonuclease.
  • kits comprising: (a) Type V endonuclease, or a nucleic acid encoding the Type V endonuclease; and (b) Type V gRNA, wherein the gRNA and the Type V endonuclease do not naturally occur together, wherein the gRNA is capable of hybridizing to a target sequence in a single stranded or double stranded target DNA, and the gRNA is capable of forming a complex with the Type II endonuclease.
  • the reagent components are provided in lyophilized form.
  • the reagent components are provided individually (either lyophilized or not lyophilized), in other embodiments, the reagent componets are provided in a pre-mixed format (either lyophilized or not lyophilized).
  • kit reagent components useful for the detection of SARS-CoV-2, a RNA virus, using one of the novel Type V or Type VI endonucleases of the disclosure.
  • Lyophilized reaction mix containing reagents and CRISPR-Cas enzyme gRNA-RNP complexes for detection of a SARS-CoV-2 amplification product.
  • Such mix may also include a labeled reporter, e.g. a 5 ’FAM-3 ’Quencher ssRNA or ssDNA-based oligonucleotide reporter, or a 5 ’FAMS’ Quencher single stranded DNA/RNA chimera-based oligonucleotide reporter.
  • RNA-RNP complexes for detection of RNAse P amplification product.
  • Such mix may also include a labeled reporter, e.g. a 5 ’FAM-3’ Quencher RNA-based oligonucleotide reporter.
  • Metagenome sequences were obtained from environmental samples, and compiled to construct a database of putative CRISPR-Cas loci.
  • CRISPR arrays were identified using CrisprCasFinder software. The criteria of filtering were putative Class II Type II, V, and VI effectors > 400 aa, which were adjacent to cas genes and CRISPR arrays. Sequences were aligned with Clustal Omega using HMM profiles. Genes were identified from metagenomic samples. Scripts were run on the sequences, designed to find CRISPR sequences and accompanying genes encoding proteins showing homology with reported Cas enzymes. Comparative BlastP analyses were performed against sequences deposited in databases (NCBI, LENS), discarding those candidates showing Id% >50 with deposited proteins. Presence of specific domains (e.g. RuvC, HEPN) and catalytic motifs were determined (CD-search, phmmer, UNIPROT). The novel endonucleases described herein were identified.
  • Expression vectors were artificially synthesized. The effector plasmid codon optimization, synthesis, and cloning were generated. Expression plasmids were transformed into E. coli.
  • SEQ ID NO: 1 represents a novel Type V variant of the disclosure, Type V Cas l, (1283 amino acids in length).
  • FIG. 4 shows the molecular weight and purity using SDS-PAGE after protein purification.
  • the Type V Cas l protein was purified via the following scheme. Recombinant protein was expressed in E. coli NiCo21 (DE3) cells (NEB # C2529H) harboring the pET28a/ Type V Cas_l-H6X expression plasmid by growing in LB broth culture medium at 37°C followed by induction of expression at 28°C for 3 hr in presence of 0.25 mM IPTG. Cells were disrupted by sonication prior to chromatographic purification.
  • Recombinant protein was purified using a HisTrapHP(Ni-NTA) (GE Healthcare) followed by a HiPrepTM 26/10desalting column (GE Healthcare) where the protein was desalted into storage buffer containing Tris-HCl 50 mM (pH 8), NaCl 200 mM, MgC12 20 mM, DTT ImM. Protein purity was controlled by Coomassie blue staining after SDS-PAGE on a 10% polyacrylamide gel. Protein concentrations were determined by UV spectroscopy and Qubit protein assay (Invitrogen). Purified proteins were stored at -80°C
  • FIG. 5 shows the results of a temperature-based assay to assess the stability of the Type V Cas l protein.
  • the first derivative plots of the melting curve diplay the thermostability of apo protein form and its binary complex (Type V Cas_l + sgRNA) .
  • the melting curve was obtained using Sypro Orange thermal shift (Invitrogen).
  • Protein melting assay was performed in a StepOneTM Real-Time PCR System (Thermo Fisher) over a temperature range from 20°C to 95°C, at a rate of l°C/minute, with 1 acquisitions/minute. The first derivative of the raw fluorescence data was taken in order to determine the Tm of the protein.
  • FIG. 6 shows the Type V Cas t trans-cleavage activity on single-stranded DNA reporter.
  • the specificity of trans-cleavage activity was tested using customized ssDNA 5’6-FAM TTATTATT- 3IABkFQ3’ from IDT (Integrated DNA Technologies, Inc) as reporter.
  • the results show that Type V Cas t is able to cleave the ssDNA reporter used.
  • the detection assay was performed at 37°C using Type V Cas_l complexes to a final concentration of 75 nM Cas : 75 nM sgRNA : 10 nM activator in a solution containing 1 x Binding Buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgC12, 1 mM DTT, 100 g/ml BSA, pH 7.9) and 600 nM of ssDNA FAMQ reporter substrate in a 40 pl reaction.
  • 1 x Binding Buffer 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgC12, 1 mM DTT, 100 g/ml BSA, pH 7.9
  • 600 nM of ssDNA FAMQ reporter substrate in a 40 pl reaction.
  • FIG. 7 shows the activity of Type V Cas t protein at different temperatures (25°C-50°C).
  • the efficiency of trans-cleavage activity at different temperatures was tested using customized ssDNA 5’6- FAM TTATTATT-3IABkFQ3’ from IDT (Integrated DNA Technologies, Inc) as a reporter.
  • the results showed that Type V Cas t is able to cleave with similar efficiency the ssDNA reporter in a wide range from room temperature even as high as 50°C.
  • the detection assay was performed at 25°C, 30°C, 38°C and 50°C using Type V Cas_l complexes to a final concentration of 75 nM Cas : 75 nM sgRNA : 10 nM activator in a solution containing 1 x Binding Buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgC12, 1 mM DTT, 100 g/ml BSA, pH 7.9) and 600 nM of ssDNA FAMQ reporter substrate in a 40 pl reaction.
  • 1 x Binding Buffer 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgC12, 1 mM DTT, 100 g/ml BSA, pH 7.9
  • 600 nM of ssDNA FAMQ reporter substrate in a 40 pl reaction.
  • FIGs. 69A-69B show collateral activity for Type V Cas t protein complex using as substrate a single-stranded DNA (IDT primer) (FIG. 69A) and (B) (FIG. 69B) double-stranded DNA (customized plasmid containing Hanta sequence).
  • IDT primer single-stranded DNA
  • B double-stranded DNA
  • the activity was measured at 37°C for Ih in presence of MnCh and/or MgCh.
  • the addition of manganese increase the speed of the reaction and is essential when using dsDNA as target.
  • the reaction was initiated by preparing complexes to a final concentration of 150 nM Type V Cas_l : 150 nM sgRNA : 10 nM activator or 10 nM of double-stranded DNA in a solution containing lx Binding Buffer (50 mM NaCl, 10 mM Tris-HCl, 1 mM DTT, 100 g/ml BSA, 10 mM of MgCh and/or lOnM MnCh, pH 7.9,).
  • the specificity of trans-cleavage activity was tested using customized ssDNA /56-FAM/TTATTATT/3IABkFQ/ from IDT (Integrated DNA Technologies, Inc.) as reporter. Control groups without Cas enzyme, guide or target were included and non-collateral cleavage was observed.
  • FIGs. 70A and 70B show trans-cleavage activities on single-stranded reporters.
  • Detection assays were performed at 37°C using Type V Cas t complex to a final concentration of 150 nM Type V Cas_l : 150 nM sgRNA : 10 nM activator in a solution containing 1 x Binding Buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgC12, 1 mM DTT, 100 g/ml BSA, pH 7.9) and 600 nM of FAMQ reporter substrates (ssRNA 5’6-FAM rArUrArUrArUrArA-3IABkFQ3, RNaseAlert (Cat N 11-04- 03-03- IDT, ssDNA (/56-FAM/TTATTATT/3IABkFQ/) and Hybrid DNA/RNA (/56- FAM/TTATrUrArUrU/3IABkFQ/) in a 40 pl reaction.
  • Binding Buffer 50 mM NaC
  • FIG. 71 shows the specific activity for dsDNA cleavage site determination. The results showed that Type V Cas t protein cuts at the 13th base site of the non-complementary strand and the 18th base site of the complementary strand downstream of the PAM sequence, generating a 5-nt overhang when the spacer length is 23 nt.
  • Type V Cas t complex was performed at 37°C using Type V Cas t complex to a final concentration of 500 nM Type V Cas_l : 500 nM sgRNA : pGEM-T easy/Hanta dsDNA, 3 pg in a solution containing 1 x Binding Buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgC12, 1 mM DTT, 100 g/ml BSA, pH 7.9). Reactions were incubated 4 hours and the product was sent to a sequencing service.
  • 1 x Binding Buffer 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgC12, 1 mM DTT, 100 g/ml BSA, pH 7.9
  • Detection assays were performed at 37°C using Type V Cas t complex to a final concentration of 150 nM Type V Cas_l : 150 nM sgRNA : 10 nM activator in a solution containing 1 x Binding Buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgC12, 1 mM DTT, 100 g/ml BSA, pH 7.9) and 600 nM of FAMQ reporter substrates (ssRNA 5’6-FAM rArUrArUrArUrArA-3IABkFQ3, RNaseAlert (Cat N 11-04-03-03- IDT, ssDNA (/56-FAM/TTATTATT/3IABkFQ/)) and Hybrid DNA/RNA (/56- FAM/TTATrUrArUrU/3IABkFQ/) in a 40 pl reaction.
  • Binding Buffer 50 mM Na
  • SEQ ID NO: 2 represents a novel Type V variant of the disclosure, Type V Cas_2, (1235 amino acids in length).
  • FIG. 8 is a schematic representation of the organization of the CRISPR Cas cluster loci around the novel Type V Cas_2 gene of the disclosure.
  • FIG. 10 shows the amino acid sequence of Type V Cas_2 with the RuvC motifs underlined/highlighted (SEQ ID NO: 2). The FnType V sequence referenced in Shmakov et al., 2015 was used as a reference for identification of the Ruv motifs.
  • FIG. 11 shows Type V Cas_2 molecular weight and purity using SDS-PAGE after protein purification.
  • Recombinant protein was expressed in E. coli Rosetta (DE3) cells (Novagen #70954) harboring the pET28a(+)-TEV/Cas expression plasmid by growing in LB broth culture medium at 37°C followed by induction of expression at 28°C for 6 hr in presence of 0.25 mM IPTG. Cells were disrupted by sonication prior to chromatographic purification.
  • Recombinant protein was purified using a HisTrapHP(Ni-NTA) (GE Healthcare) followed by a HiPrepTM 26/10 desalting column (GE Healthcare) where the protein was desalted into storage buffer containing Tris-HCl 50 mM (pH 8), NaCl 200 mM, MgC12 20 mM, DTT ImM. Protein purity was controlled by Coomassie blue staining after SDS- PAGE on a 10% polyacrylamide gel. Protein concentrations were determined by UV spectroscopy and Qubit protein assay (Invitrogen). Purified proteins were stored at -80°C
  • FIG. 12 shows that the protein Type V Cas_2 and its binary complex (Type V Cas_2 + sgRNA) are thermostable.
  • the first derivative plots of melting curve displaying the thermostability of apo protein form and binary complex.
  • the melting curve was obtained by a thermal shift assay using Sypro Orange (Invitrogen).
  • Binary complex protein + sgRNA was formed at a 1 : 1 ratio. Apo and complexes were incubated at room temperature for 10 minutes prior to melting to assure complex formation. The reactions were then split into three 20uL technical replicates.
  • Protein melting assay was performed in a StepOneTM Real-Time PCR System (Thermo Fisher) over a temperature range from 20°C to 95°C, at a rate of l°C/minute, with 1 acquisitions/minute. The first derivative of the raw fluorescence data was taken in order to determine the Tm of the protein.
  • FIG. 72 shows trans-cleavage activity testing DTT and MnCh as additives in a temperature range (46°C-60°C).
  • the efficiency of trans-cleavage activity at different temperatures was tested using customized ssDNA 5’6-FAM TTATTATT-3IABkFQ3’ from IDT (Integrated DNA Technologies, Inc.) as a reporter. High MnCh concentrations are detrimental for activity, lower concentrations were tested in a wider range of temperatures.
  • DTT was increased at 5 mM to prevent manganese oxidation. At lower temperature 2mM of MnCh presented the higher activities.
  • Detection assay was performed at 46°C, 50°C, 52.5°C and 60°C using Type V Cas_2 complexes to a final concentration of 150 nM
  • Type V Cas_2 150 nM sgRNA: 50 nM activator in a solution containing 1 x Binding Buffer (25 mM NaCl, 10 mM Tris-HCl, 10 mM MgC12, 5 mM DTT, 100 g/ml BSA, pH 8.8, MnCh 0.5, 1, 2mM) and 600 nM of ssDNA FAMQ reporter substrate in a 40 pl reaction.
  • FIG. 73 shows the activity of Type V Cas_2 protein in a temperature curve (32.8°C- 45°C). The efficiency of trans-cleavage activity at different temperatures was tested using customized ssDNA /56-FAM/TTATTATT/3IABkFQ/ from IDT (Integrated DNA Technologies, Inc.) as a reporter. The results showed that Type V Cas_2 is able to cleave with low efficiency the ssDNA reporter only between 42.8°C and 45°C.
  • Detection assay was performed at 32.8°C, 34.5°C, 37°C, 40.2°C, 42.8°C and 45°C using Type V Cas_2 complexes to a final concentration of 150 nM Type V Cas_2 : 150 nM sgRNA : 50 nM activator in a solution containing 1 x Binding Buffer (25 mM NaCl, 10 mM Tris-HCl, 10 mM MgC12, 5 mM DTT, 100 g/ml BSA, pH 8.8, 2mM MnCh) and 600 nM of ssDNA FAMQ reporter substrate in a 40 pl reaction.
  • 1 x Binding Buffer 25 mM NaCl, 10 mM Tris-HCl, 10 mM MgC12, 5 mM DTT, 100 g/ml BSA, pH 8.8, 2mM MnCh
  • FIG.74 shows differential efficiency in dinucleotide reporter cleavage. Different reporter sequences were tested showing a significant increase in Type V Cas_2 activity. This enzyme has demonstrated a highly efficiency in All Dinucleotide_A-G cleavage, evidenced by increased fluorescence in compare with ssDNA determined FAMQ TTATTATT reporter sequence.
  • Type V Cas_2 150 nM sgRNA : 10 nM ssDNA Hanta target, in a solution containing 1 x Binding Buffer (25 mM NaCl, 10 mM Tris-HCl, 10 mM MgC12, 5 mM DTT, 100 g/ml BSA, 2mM MnCh, pH 8.8) and 1.25 pM of customized FAMQ reporter substrates (/56-FAM/TTATTATT/3IABkFQ/, All Dinucleotide A- G/56 FAM/ATACAGAGTGCG/3IABkFQ/ (SEQ ID NO: 143), All Dinucleotide_CT/56- FAM/TATGTCTCACGC/3IABkFQ/ (SEQ ID NO: 144) and Poly Nucleotide All Polynucleotides 156- FAM/AAATTTCCC
  • SEQ ID NO: 7 represents a novel Type V variant of the disclosure, Type V Cas_7, (1245 amino acids in length).
  • FIG. 25 is a schematic representation of the organization of the CRISPR Cas cluster loci around the novel Type V Cas_7 gene of the disclosure.
  • FIG. 27 shows the amino acid sequence of Type V Cas_7 with the RuvC motifs underlined/highlighted (SEQ ID NO: 7). The FnType V sequence referenced in Shmakov et al., 2015 was used as a reference for identification of the Ruv motifs.
  • FIG. 28 shoes Type V Cas_7’s molecular weight and purity through SDS-PAGE.
  • the protein was purified via the following scheme. Recombinant protein was expressed in E. coli NiCo21 (DE3) cells (NEB # C2529H) harboring the pET28a/ Type V Cas_7 -H6X expression plasmid by growing in LB broth culture medium at 37°C followed by induction of expression at 28°C for 6 hr in presence of 0.25 mM IPTG. Cells were disrupted by sonication prior to chromatographic purification.
  • Recombinant protein was purified using a HisTrapHP(Ni-NTA) (GE Healthcare) followed by a HiPrepTM 26/10desalting column (GE Healthcare) where the protein was desalted into storage buffer containing Tris-HCl 50 mM (pH 8), NaCl 200 mM, MgC12 20 mM, DTT ImM. Protein purity was controlled by Coomassie blue staining after SDS-PAGE on a 10% polyacrylamide gel. Protein concentrations were determined by UV spectroscopy and Qubit protein assay (Invitrogen). Purified proteins were stored at -80°C. [0489] FIG. 29 shows the results of a temperature-based assay to assess the stability of Type V Cas_7 protein.
  • FIG. 75 shows a 10% SDS-PAGE analysis of Type V Cas_3 purification.
  • TE total extract (2 pl)
  • P Pellet (4 pl)
  • SN supernatant (4 pl)
  • FT Flow through (4 pl) NaCl: wash with E buffer (15 pl)
  • F wash with F buffer (15 pl)
  • E Elution with G buffer (8 pl)
  • D desalted protein (8 pl).
  • Storage sample of storage protein aliquots. Results are shown in FIG. 75.
  • FIG. 76 shows the results of a temperature-based assay to assess the stability of Type V Cas_3 protein.
  • FIG. 77 shows ssDNA collateral cleavage of the Type V Cas_3 protein for an exemplary ssDNA Hantavirus target.
  • a curve of pH (6.9 to 9.6), various salt concentration (25-200 mM NaCl), the addition of MnCh and three commercial buffer conditions (2.1 NEB, CutSmart NEB and Isothermal Amplification Buffer NEB) were tested.
  • the efficiency of trans-cleavage activity at different reaction buffer conditions was tested using customized ssDNA /56-FAM/TTATTATT/3IABkFQ/ from IDT (Integrated DNA Technologies, Inc) as a reporter.
  • the best activity was obtained in buffer 2.1 (New England Biotechnology), at high pH (> 8) and low salt concentrations (25-100mM).
  • the addition of manganese (2mM MnCh) to NEB 2.1 buffer does not improves the reaction.
  • Detection assay was performed at 30°C using Type V Cas_3 complexes to a final concentration of 150 nM Type V Cas_3 : 150 nM sgRNA: 10 nM activator in a solution containing 1 x Binding Buffer and 625 nM of each ssDNA FAMQ reporter substrate in a 40 pl reaction.
  • Three different commercial Binding Buffers were tested: NEB 2.1, CutSmart and Isothermal Amplification Buffer (New England Biotechnology), a curve of pH (from 6.8 to 9.6) was prepared using the base of a 2.1 NEB buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCh, 100 g/ml BSA).
  • the salt concentration curve (25-200 mM NaCl) was prepared at 7.9 pH from 2.1 NEB buffer (25-200 mM NaCl, 10 mM Tris- HCl, 10 mM MgCh, 100 g/ml BSA, pH 7.9). Reactions were incubated 120 minutes in a fluorescence plate reader Synergy Hl (Bio-Tek) and background-corrected fluorescence values were calculated by subtracting fluorescence values obtained from reactions carried out by triplicate in the absence of ssDNA Hanta target. Results are shown in FIG. 77
  • FIG. 78 shows the activity of Type V Cas_3 protein at different temperatures (30°C-50°C).
  • the efficiency of trans-cleavage activity at different temperatures was tested using customized ssDNA /56-FAM/TTATTATT/3IABkFQ/ from IDT (Integrated DNA Technologies, Inc) as a reporter.
  • the results showed that Type V Cas_3 is able to cleave the ssDNA reporter in a wide range of temperatures from 30°C to 46.5°C showing a decrease in activity at higher temperatures (48-50°C).
  • NTC Non-template negative control
  • FIG. 79 shows Trans-cleavage activities on single-stranded reporters.
  • the specificity of transcleavage activity using customized ssDNA or ssRNA as reporters was tested.
  • the results showed that Type V Cas_3 protein is able to cleave DNA or RNA reporters with different specificities.
  • Both DNA and RNA guanine homopolymers (Poly G) reporters were not cleaved by Type V Cas_3 protein and as a consequence a decreased activity was observed in dimers that contained guanine nucleotides in their composition.
  • Detection assays were performed at 40°C using Type V Cas_3 complex to a final concentration of 150 nM Type V Cas_3 : 150 nM sgRNA : 10 nM activator in a solution containing 1 x Binding Buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCh, 100 g/ml BSA, pH 7.9) and 625 nM of FAMQ reporter substrates in a 40 pl reaction. Reactions were incubated in a fluorescence plate reader Synergy Hl (Bio-Tek) and background-corrected fluorescence values were calculated by subtracting fluorescence values obtained from reactions carried out by triplicate in the absence of Hanta target.
  • FIG. 80 shows a 10% SDS-PAGE analysis of Type V Cas_4 purification.
  • the Type V Cas_4 protein was purified as recombinant protein expressed in E. coll NiCo21 (DE3) cells (NEB # C2529H) harboring the pET28a/Type V Cas_4 -H6X expression plasmid by growing in LB broth culture medium at 37°C followed by induction of expression overnight at 18°C in presence of 0.25 mM IPTG. Cells were disrupted by sonication prior to chromatographic purification.
  • Recombinant protein was purified using a His-Trap HP (Ni-NTA GE Healthcare) followed by a HiPrepTM 26/10 desalting column (GE Healthcare) where the protein was desalted into storage buffer containing Tris-HCl 50 mM (pH 8), NaCl 200 mM, MgCh 20 mM, DTT ImM. Protein purity was controlled by Coomassie blue staining after SDS-PAGE on a 10% polyacrylamide gel. Protein concentrations were determined by UV spectroscopy and Qubit protein assay (Invitrogen). Purified proteins were stored at -80°C.
  • FIG. 81 shows the results of a temperature-based assay to assess the stability of Type V Cas_4 protein.
  • FIG. 82A-82C Activity test in different reaction buffer conditions.
  • FIGs. 82A-82C shows ssDNA collateral cleavage of the Type V Cas_4 protein for an exemplary ssDNA Hantavirus target.
  • a curve of pH (6.8 to 9.5), various salt concentration (25-200 mM NaCl), the addition of MnCh and three commercial buffer conditions (2.1 NEB, CutSmart NEB and Isothermal Amplification Buffer NEB) were tested.
  • the efficiency of trans-cleavage activity at different reaction buffer conditions was tested using customized ssDNA /56-FAM/TTATTATT/3IABkFQ/ from IDT (Integrated DNA Technologies, Inc) as a reporter.
  • the best activity was obtained in buffer 2.1 (New England Biotechnology), at pH between 7.9 and 8.8.
  • High salt concentrations (100-200mM) were detrimental for Type V Cas_4protein activity.
  • the addition of manganese (2mM MnCh) to NEB 2.1 buffer does not improves the reaction.
  • Detection assay was performed at 30°C using Type V Cas_4 complexes to a final concentration of 150 nM Type V Cas_4: 150 nM sgRNA: 10 nM activator in a solution containing 1 x Binding Buffer and 625 nM of each ssDNA FAMQ reporter substrate in a 40 pl reaction.
  • Three different commercial Binding Buffers were tested: NEB 2.1, CutSmart and Isothermal Amplification Buffer (New England Biotechnology), a curve of pH (from 6.8 to 9.5) was prepared using the base of a 2.1 NEB buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCh, 100 g/ml BSA).
  • the salt concentration curve (25-200 mM NaCl) was prepared at 7.9 pH from 2.1 NEB buffer (25-200 mM NaCl, 10 mM Tris- HCl, 10 mM MgCh, 100 g/ml BSA, pH 7.9). Reactions were incubated 150 minutes in a fluorescence plate reader Synergy Hl (Bio-Tek) and background-corrected fluorescence values were calculated by subtracting fluorescence values obtained from reactions carried out by triplicate in the absence of ssDNA Hanta target.
  • FIG.83 shows the activity of Type V Cas_4 protein at different temperatures (30°C-50°C).
  • the efficiency of trans-cleavage activity at different temperatures was tested using customized ssDNA 156- FAM/TTATTATT/3IABkFQ/ from IDT (Integrated DNA Technologies, Inc) as a reporter.
  • the results showed that Type V Cas_4 is able to cleave the ssDNA reporter in a wide range of temperatures from 30°C to 37.6° C showing a decrease in activity at higher temperatures (>42.5°C).
  • the detection assay was performed from 30°C to 50°C using Type V Cas_4 complexes to a final concentration of 150 nM Cas : 150 nM sgRNA : 10 nM activator in a solution containing 1 x Binding Buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCh, 100 g/ml BSA, pH 7.9) and 625 nM of ssDNA FAMQ reporter substrate in a 40 pl reaction.
  • 1 x Binding Buffer 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCh, 100 g/ml BSA, pH 7.9
  • 625 nM of ssDNA FAMQ reporter substrate in a 40 pl reaction.
  • FIG. 84A-84B shows trans-cleavage activities on single-stranded reporters. We tested the specificity of trans-cleavage activity using customized ssDNA or ssRNA as reporters.
  • Type V Cas_4 protein is able to cleave DNA reporters with different specificities but not the RNA reporters tested. Moreover, DNA guanine homopolymers (Poly G) reporter were not cleaved by Type V Cas_4 protein while DNA cytokine homopolymer (Poly C) reporter and their respective dimeric variants showed the best cleavage values.
  • Detection assays were performed at 35°C using Type V Cas_4 complex to a final concentration of 150 nM Type V Cas_3 1 : 150 nM sgRNA : 10 nM activator in a solution containing 1 x Binding Buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCh, 100 g/ml BSA, pH 7.9) and 625 nM of FAMQ reporter substrates in a 40 pl reaction. Reactions were incubated in a fluorescence plate reader Synergy Hl (Bio-Tek) and background-corrected fluorescence values were calculated by subtracting fluorescence values obtained from reactions carried out by triplicate in the absence of Hanta target. Results are shown in FIGs. 84A-84B.
  • FIG. 85 shows Type V Cas_5 purification and FIG. 86 shows thermal shift analysis.
  • Type V Cas_5 protein was purified using Ni-NTA agarose chromatography. The thermal stability of the purified protein was tested using SYPRO® Orange Protein Gel Stain (Merck) as denaturalization reporter. The melting curve observed indicates that the protein is stable up to 36°C in absence of scout and sgRNA.
  • Type V Cas_5 protein coding sequence was codon-optimized and synthesized by GeneScript and then cloned into pET28a (Novagen) with N-terminal 6xHis tagging (SEQ ID NO: 146). Expression plasmids were transformed into E.
  • coli NiCo21 (DE3) (NEB).
  • NEB coli NiCo21
  • cells were grown with shaking at 200 rpm and 37 °C until the OD 600 reached 0.68, and IPTG was then added to a final concentration of 0.25 mM followed by further culture of the cells at 28 °C for about 6 h before the cell harvesting.
  • Cells were resuspended in 10 mL of buffer A (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 1 mM DTT and 10% glycerol) with protease inhibitor cocktail (Promega), 10 mM imidazole and 0,1 mg/ml lysozyme.
  • buffer A 50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 1 mM DTT and 10% glycerol
  • protease inhibitor cocktail Promega
  • Thermal stability assay was performed at a temperature range from 20°C to 90°C using 15 ug of Type V Cas_5 protein in a solution containing 1 x Desalting buffer desalting buffer (50 mM Tris-HCl pH 8, 200 mM NaCl, 20mM MgC12, 1 mM DTT) and 10 x of SYPRO® dye in a 30 pl reaction.
  • FIG. 87 shows trans-cleavage activity testing using two different sgRNA and three buffer conditions. The efficiency of trans-cleavage activity on each condition was tested using customized ssDNA /56-FAM/TTATTATT/3IABkFQ/ and ssDNA /56-FAM/NNNNNN/3IABkFQ/ from IDT (Integrated DNA Technologies, Inc.) as a reporter. 18 nucleotides sgRNA presents higher activity than 24 nucleotides sgRNA. The best activity was observed when in NEB 2.1 supplemented with ImM DTT.
  • Type V Cas_5 complexes to a final concentration of 250 nM Type V Cas_5: 250 nM scoutRNA : 250 nM sgRNA: 50 nM activator in a solution containing 1 x Binding Buffer and 625 nM of each ssDNA FAMQ reporter substrate in a 40 pl reaction.
  • Binding Buffers were tested: B 6.8 (50 mM Tris pH 6.8, 100 mM NaCl, 10 mM MgCl, 1 mM DTT), NEB 2.1+DTT (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgC12, 100 ug/ml BSA, pH 7.9, 1 mM DTT) and NEB 3.0 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgC12, pH 7.9, 1 mM DTT). A no-enzyme control was added using the 18 nucleotides sgRNA in NEB 2.1+DTT buffer.
  • FIG. 88 shows the activity of Type V Cas_5 protein in a temperature curve (52°C- 60°C) and three buffer conditions. The enzyme was incubated 20 minutes at the reported temperatures before activation with ssDNA Hanta target. The efficiency of trans-cleavage activity on each condition was tested using customized FAM/TTATTATT/3IABkFQ/ and ssDNA /56-FAM/NNNNNN/3IABkFQ/ from IDT (Integrated DNA Technologies, Inc.) as a reporter. The results showed that Type V Cas_5 is able to cleave with good efficiency the ssDNA reporters between 52°C and 56°C. The best activity was observed in buffer with pH 8.8 and 25mM NaCl.
  • Detection assay was performed at 52°C, 54°C, 56°C, 58,4°C and 60,3 °C using Type V Cas_5 complexes to a final concentration of 125 nM Type V Cas_5 : 125 nM scoutRNA : 125 nM sgRNA: 25 nM activator in a solution containing 1 x Binding Buffer and 625 nM of each ssDNA FAMQ reporter substrate in a 40 pl reaction.
  • NEB 2.1+DTT Tris 10 mM pH 7.9/NaCl 50 mM/MgCl 10 mM/BSA 100 ug/mL/DTT ImM
  • pH_8.8 Tris 10 mM pH 8.8/NaCl 50 mM/MgCl 10 mM/BSA 100 ug/mL/DTT ImM
  • pH_8.8_NaCl_25nM_MnCl_2nM Tris 10 mM pH 8.8/NaCl 25 mM/MgCl 10 mM/BSA 100 ug/mL/DTT ImM/MnCl 2nM.
  • FIGs.89A-89B are a PAM selectivity test.
  • the Type V Cas_5 activation on different left-PAM sequences was tested using short dsDNA molecules (146 bp) as targets and customized 156- FAM/TTATTATT/3IABkFQ/ and ssDNA /56-FAM/NNNNNN/3IABkFQ/ from IDT (Integrated DNA Technologies, Inc.) as reporters respectively.
  • the results showed that Type V Cas_5 is activated whit more efficiency when TC or TT PAM sequences.
  • TA PAM sequence target present a reduce activity compared to TC or TT and the less activity is observed with TG PAM sequence.
  • Detection assay was performed at 54°C Type V Cas_5 complexes to a final concentration of 125 nM Type V Cas_5: 125 nM scoutRNA : 125 nM sgRNA: 10 nM target in a solution containing 1 x Binding Buffer (Tris 10 mM pH 8.8/NaCl 25 mM/MgCl 10 mM/MnCl 2mM/BSA 100 ug/mL/DTT ImM) and 625 nM of each ssDNA FAMQ reporter substrate in a 40 pl reaction.
  • Binding Buffer Tris 10 mM pH 8.8/NaCl 25 mM/MgCl 10 mM/MnCl 2mM/BSA 100 ug/mL/DTT ImM
  • FIG.90 shows the results of the differential efficiency in dinucleotide single-stranded reporter cleavage. Different dinucleotide reporter sequences were tested showing a significant increase in Type V Cas_5 activity. This enzyme has demonstrated a highly efficiency in All Dinucleotide_A-G cleavage, evidenced by increased fluorescence in compare with ssDNA determined FAMQ TTATTATT reporter sequence.
  • Detection assay was performed at 52°C Type V Cas_5 complexes to a final concentration of 125 nM Type V Cas_5: 125 nM scoutRNA : 125 nM sgRNA: 10 nM ssDNA Hanta target in a solution containing 1 x Binding Buffer (Tris 10 mM pH 8.8, NaCl 25 mM, MgCh 10 mM, MnCh 2mM, BSA 100 ug/mL and DTT ImM) and 625 nM of customized FAMQ reporter substrates (/56- FAM/TTATTATT/3IABkFQ/, All Dinucleotide_A-G/56 FAM/ATACAGAGTGCG/3IABkFQ/ (SEQ ID NO: 143), All Dinucleotide_CT/56-FAM/TATGTCTCACGC/3IABkFQ/ (SEQ ID NO: 144) and All Polynucleotides /56-
  • FIG.91 shows the results from a differential efficiency in single-base DNA reporter cleavage. Different reporters with only one base in their sequences were tested in Type V Cas_5 activity. This enzyme has demonstrated that single base reporter sequences are cleaved with less efficiency that mixed bases reporter sequences. Among single base reporters, poly-A is cleaved with the highest efficiency followed by poly-C and poly-T. No cleavage was observed in Poly-G reporter.
  • Detection assay was performed at 54°C Type V Cas_5 complexes to a final concentration of 125 nM Type V Cas_5: 125 nM scoutRNA : 125 nM sgRNA: 10 nM ssDNA Hanta target in a solution containing 1 x Binding Buffer (Tris 10 mM pH 8.8, NaCl 25 mM, MgCh 10 mM, MnCh 2mM, BSA 100 ug/mL, DTT ImM) and 625 nM of customized FAMQ reporter substrates (All Polynucleotides 156- FAM/AAATTTCCCGGG/3IABkFQ/ (SEQ ID NO: 145) (12nt), Poly C 156- FAM/CCCCCCC/3IABkFQ/, Poly A /56-FAM/AAAAAAA/3IABkFQ/, Poly T /56-FAM/TTTTTTT /3IABkFQ/ and Poly G
  • FIGs. 92A-92B shows the results of the collateral activity of Type VI Cas_2 protein complex in different buffer solutions.
  • the efficiency of trans-cleavage activity of Type VI Cas_2 protein was tested in different buffer solutions using customized ssRNA /56-FAM/rUrUrUrUrUrUrUrUrUrU/3IABkFQ/ from IDT (Integrated DNA Technologies, Inc.) as a reporter.
  • FIG. 92A Shows the time course cleavage over 3 h in: 1.
  • CutSmart buffer from NEB 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 pg/ml BSA, pH 7.9); 2. Multicore buffer from Promega (25 mM Tris-acetate, 100 mM Potassium Acetate, 10 mM Magnesium Acetate, 1 mM DTT, pH 7.5); 3. NEB 1.1 buffer from NEB (10 mM Bis-Tris-Propane-HCl, 10 mM MgCh, 100 pg/ml BSA, pH 7); 4. Goot 1 buffer (20 mM HEPES, 60 mM NaCl, 6 mM MgCh, pH 6.8); 5.
  • Goot 1 buffer Goot 2 buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgC12, pH 7.3); Goot 1 buffer supplemented with 2 mM DTT; Smargon buffer; PBS buffer; PBS buffer supplemented with 1 mM DTT and 10 mM MgCh; NEB 2 buffer from NEB (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgC12, ImM DTT, pH 7.9); NEB 2.1 buffer from NEB (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgC12, 100 pg/ml BSA, pH 7.9); NEB 4 buffer from NEB (50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 1 mM DTT, pH 7.9); CutSmart buffer;
  • CutSmart buffer demonstrated the best activity, evidenced for the highest fluorescence values.
  • the protein also showed high activity values in NEB 4 and Multicore buffers which share similar composition to CutSmart buffer.
  • the reaction was initiated by preparing complexes to a final concentration of 150 nM Type VI Cas_2: 75 nM sgRNA : 20 nM activator (31 nt. ssRNA from Synthego) and 150 nM of ssRNA FAMQ reporter substrate in a 40 pl reaction, in each of the aforementioned buffer solutions at 37°C.
  • FIGs. 93 A-93B shows collateral activity of the Type VI Cas_2 protein complex in a temperature curve (30°C- 50°C).
  • the efficiency of trans-cleavage activity at different temperatures was tested using customized ssRNA /56-FAM/rUrUrUrUrUrUrUrUrUrU/3IABkFQ/ from IDT (Integrated DNA Technologies, Inc.) as a reporter.
  • the temperatures analyzed over time were: 37.0°C, 37.8°C, 39.5°C, 42°C, 45.2°C, 47.8°C, 49.2°C and 50°.
  • the temperatures analyzed as endpoint after 180 min (FIG.
  • Type VI Cas_2 was able to cleave the ssRNA reporter efficiently between 30°C and 42°C, with an optimal activity at 31 ,4°C.
  • Type VI Cas_2 complexes to a final concentration of 150 nM Type VI Cas_2: 75 nM sgRNA : 20 nM activator (31 nt. ssRNA from Synthego) in a solution containing 1 x Binding Buffer (50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 pg/ml BSA, pH 7.9) and 150 nM of ssRNA FAMQ reporter substrate in a 40 pl reaction.
  • Binding Buffer 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 pg/ml BSA, pH 7.9
  • NTC fluorescence values were calculated from reactions carried out in the absence of ssRNA Hanta target.
  • FIG. 94 shows the results of a 10% SDS-PAGE analysis of Type VI Cas_2 purification.
  • the Type VI Cas_2 protein was purified as recombinant protein expressed in E. coll Rosetta (DE3) cells (Merck # 70954) harboring the pET28a/Type VI Cas_2-H6X expression plasmid by growing in LB broth culture medium at 37°C followed by induction of 6 hs expression at 20°C in presence of 0.25 mM IPTG. Cells were disrupted by sonication prior to chromatographic purification.
  • Recombinant protein was purified using a His-Trap HP (Ni-NTA GE Healthcare) followed by a HiPrepTM 26/10 desalting column (GE Healthcare) where the protein was desalted into storage buffer containing 10 mM HEPES, 500 mM NaCl, 1 mM DTT, pH 7.5. Protein purity was controlled by Coomassie blue staining after SDS-PAGE on a 10% polyacrylamide gel. Protein concentrations were determined by UV spectroscopy and Qubit protein assay (Invitrogen). Purified proteins were stored at -80°C.
  • FIGs. 95A-95B Collateral activity of the Type VI Cas_2 protein complex for a ssRNA target with variable protospacer flanking sequences (PFS).
  • PFS protospacer flanking sequences
  • the different PFS present in the target comprised the 5’ sequences: AAAUUAA, AAAUCCC, AAAUUAU, AAAUAGA, AAAUACU, AAAUAAG, AUUAAUU and 3’ sequences: GAAAAAU, CGGAAAU, UAAAAAU, AAAAAAU, AUAAAAU, UAUAAAU, GAUAAAU, AAUAAAU, UUUAAAU, UAUAGUU.
  • Type VI Cas_2 was able to cleave all the targets tested with similar efficiency.
  • the target with flanking sequence 5 'AAAUAGA and 3’ GAUAAAU reported the lowest fluorescence value followed by the target with flanking sequence 5' AAAUCCC and 3’ CGGAAAU.
  • the 75-nt. target displayed higher fluorescence than the 45-nt. target.
  • Binding Buffer 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 pg/ml BSA, pH 7.9
  • FIG. 96 shows the collateral activity of the Type VI Cas_2 protein complex for different customized ssRNA reporter substrates. Efficiency of trans-cleavage activity for different customized ssRNA reporters from IDT (Integrated DNA Technologies, Inc.).
  • the ssRNA reporters analyzed were: poly A (/56-FAM/rArArArArArArArArA/3IABkFQ/), poly U (/56-FAM/rUrUrUrUrUrUrUrUrU/3IABkFQ/), dinucleotide (/56-FAM/rArUrArUrArUrArUrA/3IABkFQ/), random (/56- FAM/rUrNrNrNrNrNrN/3IABkFQ/), determined (/56-FAM/rUrUrArUrUrArUrUrU/3IABkFQ/) and RNaseAlertTM substrate from IDT.
  • Type VI Cas_2 cut poly U ssRNA reporter with the maximum efficiency followed by the determined ssRNA reporter.
  • Type VI Cas_2 complex was not able to cut poly A ssRNA reporter nor dinucleotide ssRNA reporter.
  • FIGs. 97A-97B shows the collateral activity for Type VI Cas_2 protein complexes using ssRNA and ssDNA substrates.
  • FIGs.97A-97B shows collateral activity for Type VI Cas_2 protein complex using as specific targets single-stranded RNA (IDT primer) and (B) single-stranded DNA (IDT primer).
  • trans-cleavage activity for ssRNA or ssDNA was tested using customized ssRNA (/56-FAM/rUrUrUrUrUrUrUrU/3IABkFQ/ for Type VI Cas_2 and 156- FAM/rArArArArArA/3IABkFQ/ for Psm control) and customized ssDNA FAM/AAATTTCCCGGG/3IABkFQ (SEQ ID NO: 145), FAM/ATACAGAGTGCG/3IABkFQ (SEQ ID NO: 143), FAM/TATGTCTCACGC/3IABkFQ (SEQ ID NO: 144) from IDT (Integrated DNA Technologies, Inc.) as reporters.
  • Type VI Cas_2 was able to cut ssRNA reporter but not ssDNA reporter when using ssRNA as target.
  • Type VI Cas_2 was able to cut a little of ssRNA reporter after 3 h but not ssDNA reporter when using ssDNA as target.
  • the reaction was initiated by preparing complexes to a final concentration of 100 nM Type VI Cas_2: 75 nM sgRNA : 10 nM ssRNA (75 nt.) or ssDNA (60 nt.) activator in a solution containing 1 x Binding Buffer (50 mM NaCl, 10 mM Tris-HCl, 1 mM DTT, 100 g/ml BSA, 10 mM of MgCh and/or lOnM MnCh, pH 7.9) and 250 nM ssRNA or ssDNA FAMQ reporter substrates in 40 pL reaction volume.
  • 1 x Binding Buffer 50 mM NaCl, 10 mM Tris-HCl, 1 mM DTT, 100 g/ml BSA, 10 mM of MgCh and/or lOnM MnCh, pH 7.9
  • 1 x Binding Buffer 50 mM NaCl, 10 mM Tri
  • FIG. 98 Collateral activity of Type VI Cas_4 protein complex in different buffer solutions.
  • Type VI Cas_4 protein The efficiency of trans-cleavage activity of Type VI Cas_4 protein was tested in different buffer solutions using RNaseAlertTM substrate from IDT (Integrated DNA Technologies, Inc.) as a reporter.
  • NEB 2 buffer from NEB (10 mM Tris-HCl, 50 mMNaCl, 10 mM MgC12, ImM DTT, pH 7.9); 7. Goot 2 buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgC12, pH 7.3) and 8. Goot 1 buffer (20 mM HEPES, 60 mM NaCl, 6 mM MgCh, pH 6.8).
  • Reaction in CutSmart buffer demonstrated the best activity, evidenced for the highest fluorescence values.
  • the protein also showed activity in NEB 4, Multicore, NEB 1.1 and NEB 2.1 buffers and to a lesser extent in NEB 2 buffer.
  • FIG. 99 shows the results from the collateral activity of the Type VI Cas_4 protein complex for different customized ssRNA reporter substrates. Efficiency of trans-cleavage activity for different customized ssRNA reporters from IDT (Integrated DNA Technologies, Inc.).
  • the ssRNA reporters analyzed were: poly A (/56-FAM/rArArArArArArArA/3IABkFQ/), poly U (/56- FAM/rUrUrUrUrUrUrUrUrU/3IABkFQ/), random (/56-FAM/rUrNrNrNrNrNrNrN/3IABkFQ/), determined (/56-FAM/rUrUrArUrUrArUrArUrU/3IABkFQ/) and RNaseAlert substrate from IDT.
  • Type VI Cas_4 was able to cut all the reporter substrates tested, with a higher preference for RNaseAlert, followed by the determined and poly U ssRNA reporters.
  • Experiments were performed in 40 pL reaction volume containing 1 x Binding Buffer (50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 pg/ml BSA, pH 7.9), Type VI Cas_4 protein complexed to a final concentration of 250 nM Type VI Cas_4: 125 nM sgRNA : 20 nM activator (75 nt.
  • Binding Buffer 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 pg/ml BSA, pH 7.9
  • ssRNA FQ substrates kex: 485 nm; kern: 538 nm.
  • NTC Non-template negative control
  • FIG. 100 shows 10% SDS-PAGE analysis of Type VI Cas_2 purification.
  • the Type VI Cas_4 protein was purified as recombinant protein expressed in E. coll NiCo21 (DE3) cells (NEB # C2529H) harboring the pET28a/ Type VI Cas_4-H6X expression plasmid by growing in LB broth culture medium at 37°C followed by induction of expression overnight at 24°C in presence of 0.25 mM IPTG. Cells were disrupted by sonication prior to chromatographic purification.
  • Recombinant protein was purified using a His-Trap HP (Ni-NTA GE Healthcare) followed by a HiPrepTM 26/10 desalting column (GE Healthcare) where the protein was desalted into storage buffer containing 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM DTT and 20mM MgCl 2 . Protein purity was controlled by Coomassie blue staining after SDS-PAGE on a 10% polyacrylamide gel. Protein concentrations were determined by UV spectroscopy and Qubit protein assay (Invitrogen). Purified proteins were stored at -80°C.
  • FIG. 101 shows collateral activity of the Type VI Cas_4 protein complex in a temperature curve (30°C- 50°C).
  • the efficiency of trans-cleavage activity at different temperatures was tested using RNaseAlertTM substrate from IDT (Integrated DNA Technologies, Inc.) as a reporter.
  • the temperatures analyzed in a time course cleavage were: 30.0°C, 31.2°C, 33.8°C, 37.6°C, 42.5°C, 46.5°C, 48.8°C and 50.0°.
  • the results showed that Type VI Cas_4 was able to cleave the ssRNA reporter more efficiently in the range between 30-42.5 °C, with an optimal activity at 33.8°C.
  • Type VI Cas_4 complexes to a final concentration of 250 nM Type VI Cas_4: 125 nM sgRNA : 20 nM activator (75 nt. ssRNA from Synthego) in a solution containing 1 x Binding Buffer (50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 pg/ml BSA, pH 7.9) and 150 nM of ssRNA FAMQ reporter substrate in a 40 pl reaction.
  • Binding Buffer 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 pg/ml BSA, pH 7.9
  • 150 nM of ssRNA FAMQ reporter substrate in a 40 pl reaction.
  • FIG.102 depicts the collateral activity for Type VI Cas_4protein complex using ssRNA and ssDNA substrates. Collateral activity for Type VI Cas_4 protein complex using as specific targets single-stranded RNA (IDT primer) and (B) single-stranded DNA (Macrogen primer).
  • the reaction was initiated by preparing complexes to a final concentration of 250 nM Type VI Cas_4: 125 nM sgRNA: 10 nM ssRNA (75 nt.) or ssDNA (60 nt.) activator in a solution containing 1 x Binding Buffer (50 mM NaCl, 10 mM Tris-HCl, 1 mM DTT, 100 g/ml BSA, 10 mM of MgCh and/or lOnM MnCh, pH 7.9) and 250 nM ssRNA or ssDNA FAMQ reporter substrates in 40 pL reaction volume.
  • 1 x Binding Buffer 50 mM NaCl, 10 mM Tris-HCl, 1 mM DTT, 100 g/ml BSA, 10 mM of MgCh and/or lOnM MnCh, pH 7.9
  • 250 nM ssRNA or ssDNA FAMQ reporter substrates in 40
  • NTC fluorescence values were calculated from reactions carried out in the absence of ssRNA Hanta target.

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