WO2024078645A2 - Protéine cas et son utilisation - Google Patents

Protéine cas et son utilisation Download PDF

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Publication number
WO2024078645A2
WO2024078645A2 PCT/CN2023/142927 CN2023142927W WO2024078645A2 WO 2024078645 A2 WO2024078645 A2 WO 2024078645A2 CN 2023142927 W CN2023142927 W CN 2023142927W WO 2024078645 A2 WO2024078645 A2 WO 2024078645A2
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WO
WIPO (PCT)
Prior art keywords
cas protein
sequence
disease
present
specific embodiment
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
PCT/CN2023/142927
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English (en)
Chinese (zh)
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WO2024078645A3 (fr
Inventor
梁峻彬
陈重建
孙阳
潘伟业
司凯威
黄连成
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang Synsorbio Technology Co Ltd
Guangzhou Reforgene Medicine Co Ltd
Original Assignee
Zhejiang Synsorbio Technology Co Ltd
Guangzhou Reforgene Medicine Co Ltd
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Publication date
Application filed by Zhejiang Synsorbio Technology Co Ltd, Guangzhou Reforgene Medicine Co Ltd filed Critical Zhejiang Synsorbio Technology Co Ltd
Priority to PCT/CN2023/142927 priority Critical patent/WO2024078645A2/fr
Publication of WO2024078645A2 publication Critical patent/WO2024078645A2/fr
Publication of WO2024078645A3 publication Critical patent/WO2024078645A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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 [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present disclosure relates to the field of CRISPR gene editing, and specifically to Cas proteins and their applications.
  • the CRISPR-Cas system is an adaptive immune defense formed by bacteria and archaea during the long evolution process, which can be used to fight against invading viruses and foreign DNA.
  • the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein system (CRISPR-Cas system) can directly change, modify or regulate gene sequences in cells, which is a fast and effective method.
  • the present invention provides Cas proteins and applications thereof.
  • a technical solution provided by the present invention is: a Cas protein, the amino acid sequence of which comprises or is an amino acid sequence that is at least 50% identical to any one of SEQ ID NO: 1-461.
  • the sequence table lists the Cas proteins with sequences of SEQ ID NO: 1-461, the direct repeat sequences corresponding to all Cas proteins (SEQ ID NO: 462-922), and the tracrRNA sequences corresponding to some Cas proteins (SEQ ID NO: 923-1183).
  • the at least 50% identity is 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity.
  • the amino acid sequence of the Cas protein comprises or is an amino acid sequence having 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identity to any one of SEQ ID NOs: 1-461.
  • the amino acid sequence of the Cas protein comprises or is an amino acid sequence that is at least 80% identical to any one of SEQ ID NO: 1-461.
  • the amino acid sequence of the Cas protein comprises or is an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 1-461.
  • the amino acid sequence of the Cas protein comprises or is an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 1-461.
  • the amino acid sequence of the Cas protein comprises or is an amino acid sequence that is at least 95% identical to any one of SEQ ID NO: 1-461.
  • the amino acid sequence of the Cas protein comprises or is an amino acid sequence that is at least 97% identical to any one of SEQ ID NO: 1-461.
  • the amino acid sequence of the Cas protein comprises or is an amino acid sequence that is at least 98% identical to any one of SEQ ID NO: 1-461.
  • the amino acid sequence of the Cas protein comprises or is an amino acid sequence that is at least 99% identical to any one of SEQ ID NO: 1-461.
  • the amino acid sequence of the Cas protein comprises or is an amino acid sequence that is at least 99.5% identical to any one of SEQ ID NO: 1-461.
  • the amino acid sequence of the Cas protein comprises or is an amino acid sequence that is at least 99.7% identical to any one of SEQ ID NO: 1-461.
  • the amino acid sequence of the Cas protein comprises or is an amino acid sequence that is at least 99.8% identical to any one of SEQ ID NO: 1-461.
  • the amino acid sequence of the Cas protein comprises or is an amino acid sequence that is 100% identical to any one of SEQ ID NO: 1-461.
  • the Cas protein retains the function of the protein shown in any sequence of SEQ ID NO: 1-461.
  • the Cas protein can form a complex with a guide polynucleotide. In a specific embodiment of the present invention, the Cas protein can specifically bind to a target nucleic acid with a guide polynucleotide.
  • the Cas protein can form a complex with a guide polynucleotide, and the complex can specifically bind to a target nucleic acid.
  • the Cas protein can form a complex with a guide polynucleotide, and the complex can specifically bind to a target DNA.
  • the Cas protein can specifically bind to the guide polynucleotide and cut the target Nucleic acid. In a specific embodiment of the present invention, the Cas protein can specifically bind to the guide polynucleotide and cut the target DNA. In a specific embodiment of the present invention, the Cas protein can form a complex with the guide polynucleotide, and the complex can specifically bind to and cut the target nucleic acid. In a specific embodiment of the present invention, the Cas protein can form a complex with the guide polynucleotide, and the complex can specifically bind to and cut the target DNA.
  • the retention of the function of the protein shown in any one of the sequences of SEQ ID NO: 1-461 refers to retaining the ability to form a complex with a guide polynucleotide, retaining the ability to bind to a target nucleic acid complementary to the guide sequence of the guide polynucleotide, retaining the ability to target and cut the target nucleic acid with the guide polynucleotide, and/or retaining the ability to process an RNA transcript containing a guide sequence into a guide polynucleotide molecule.
  • the function of retaining the protein shown in any one of the sequences as SEQ ID NO: 1-461 is to retain the ability to form a complex with the guiding polynucleotide.
  • the function of retaining the protein shown in any one of the sequences as SEQ ID NO: 1-461 is to retain the ability to bind to the target nucleic acid complementary to the guide sequence of the guide polynucleotide.
  • the function of retaining the protein shown in any sequence of SEQ ID NO: 1-461 is to retain and guide the ability of polynucleotides to targetedly cut the target nucleic acid.
  • the function of retaining the protein shown in any one of the sequences as SEQ ID NO: 1-461 is to retain the ability to process the RNA transcript containing the guide sequence into a guide polynucleotide molecule.
  • the amino acid sequence of the Cas protein comprises or is an amino acid sequence as shown in any one of SEQ ID NO: 1-461.
  • the PAM sequence (5' ⁇ 3') recognizable by the Cas protein is selected from any one or more of the following:
  • the N is A, T, C or G.
  • the Cas protein can recognize a PAM with a sequence of A.
  • the Cas protein can recognize a PAM with a sequence of C.
  • the Cas protein can recognize a PAM with a sequence of T.
  • the Cas protein can recognize a PAM with a sequence of G.
  • the Cas protein can recognize a PAM with a sequence of 5'-TA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GANC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GACN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GATC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AANA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NATG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CACT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AATG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NATC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GACT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NANC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GACC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AACG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AATC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AATN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TACT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GANT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CANC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TANN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GANA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CACA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CANA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CACG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TACA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AANT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TATG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TANT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NACN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NANT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TACN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NANA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AANG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NATN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CATN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CACC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AATA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CACN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GANG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CANN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GATG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NANG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NACG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NANN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TACC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CATC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GACA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GATA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AANC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TANC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TACG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CANT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TATN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CATA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NACA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NACC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TANG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CATT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NATT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TATT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GANN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGCT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CATG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GATT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AACN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AACT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TAAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TNTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AACC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GTTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TATA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NCGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GACG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TATC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AANN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGNN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TANA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCAA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGNG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NATA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GATN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNTA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTTT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AGGN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ACNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NTGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NACT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NAAC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GGNT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CCCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ANGA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCTC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTCG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AATT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCGC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-ATAG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAAN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AACA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CAGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GNNA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TGCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GCGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGGG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CANG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TTTG-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-GAGT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-AAAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CTCA-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNCN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CNCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-TCTN-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGNC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NGCC-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-CGAT-3'.
  • the Cas protein can recognize a PAM with a sequence of 5'-NNGC-3'.
  • the Cas protein is an inactivated variant of the Cas protein.
  • the inactivated variant of the Cas protein is a dead Cas protein or a nickase Cas protein.
  • the Cas protein is selected from the active fragments constituting any one of the Cas proteins of the present invention.
  • a technical solution provided by the present invention is: a guiding polynucleotide, which comprises (i) a direct repeat sequence, wherein the direct repeat sequence has at least 50% identity with any one of SEQ ID NO: 462-922, and (ii) a guiding sequence engineered to hybridize with a target nucleic acid; the direct repeat sequence is connected to the guiding sequence, and the guiding polynucleotide is capable of forming a complex with the Cas protein and guiding the sequence-specific binding of the complex to the target nucleic acid.
  • the homologous repeated sequence has 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to any one of SEQ ID NO:462-922.
  • the same direction repeating sequence has at least 60% sequence identity compared to any one of SEQ ID NO: 462-922. In some embodiments of the invention, the same direction repeating sequence has at least 65% sequence identity compared to any one of SEQ ID NO: 462-922. In some embodiments of the invention, the same direction repeating sequence has at least 70% sequence identity compared to any one of SEQ ID NO: 462-922. In some embodiments of the invention, the same direction repeating sequence has at least 75% sequence identity compared to any one of SEQ ID NO: 462-922. In some embodiments of the invention, the same direction repeating sequence has at least 80% sequence identity compared to any one of SEQ ID NO: 462-922.
  • the same direction repeating sequence has at least 85% sequence identity compared to any one of SEQ ID NO: 462-922. In some embodiments of the invention, the same direction repeating sequence has at least 90% sequence identity compared to any one of SEQ ID NO: 462-922. In some embodiments of the invention, the same direction repeating sequence has at least 95% sequence identity compared to any one of SEQ ID NO: 462-922. In some embodiments of the invention, the same direction repeating sequence has at least 96% sequence identity compared to any one of SEQ ID NO: 462-922. In some embodiments of the invention, the same direction repeating sequence has at least 97% sequence identity compared to any one of SEQ ID NO: 462-922.
  • the same direction repeating sequence has at least 98% sequence identity compared to any one of SEQ ID NO: 462-922. In some embodiments of the invention, the same direction repeating sequence has 100% sequence identity compared to any one of SEQ ID NO: 462-922.
  • the Cas protein is the Cas protein described in the present invention.
  • the guide sequence comprises 15-60 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-50 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-40 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-35 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-30 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-25 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 18-25 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 20-25 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 18-22 nucleotides.
  • the guide sequence comprises 20-22 nucleotides. In specific embodiments of the invention, the guide sequence comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.
  • the guide sequence hybridizes to the target nucleic acid, and the guide sequence is 90%-100% complementary to the target nucleic acid.
  • said guide sequence hybridizes to said target nucleic acid.
  • the guide sequence hybridizes to the target nucleic acid, and the guide sequence is mismatched with the target nucleic acid by no more than one nucleotide.
  • the direct repeat sequence comprises 15-100 nucleotides. In a specific embodiment of the invention, the direct repeat sequence comprises 15-90 nucleotides. In a specific embodiment of the invention, the direct repeat sequence comprises 15-80 nucleotides. In a specific embodiment of the invention, the direct repeat sequence comprises 15-70 nucleotides. In a specific embodiment of the invention, the direct repeat sequence comprises 15-60 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-50 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-40 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 20-40 nucleotides.
  • the guide sequence comprises 20-30 nucleotides. In specific embodiments of the invention, the guide sequence comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides.
  • the guide sequence is located at the 3' end of the direct repeat sequence.
  • the guide sequence is located at the 5' end of the direct repeat sequence.
  • the guide polynucleotide further comprises tracrRNA.
  • the tracrRNA sequence has at least 50% identity to any one of SEQ ID NOs: 923-1183. In some embodiments of the invention, the tracrRNA sequence has 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 923-1183.
  • the tracrRNA sequence has at least 60% sequence identity compared to any one of SEQ ID NO: 923-1183. In some embodiments of the invention, the tracrRNA sequence has at least 65% sequence identity compared to any one of SEQ ID NO: 923-1183. In some embodiments of the invention, the tracrRNA sequence has at least 70% sequence identity compared to any one of SEQ ID NO: 923-1183. In some embodiments of the invention, the tracrRNA sequence has at least 75% sequence identity compared to any one of SEQ ID NO: 923-1183. In some embodiments of the invention, the tracrRNA sequence has at least 80% sequence identity compared to any one of SEQ ID NO: 923-1183.
  • the tracrRNA sequence has at least 85% sequence identity compared to any one of SEQ ID NO: 923-1183. In some embodiments of the invention, the tracrRNA sequence has at least 90% sequence identity compared to any one of SEQ ID NO: 923-1183. In some embodiments of the invention, the tracrRNA sequence has at least 95% sequence identity compared to any one of SEQ ID NO: 923-1183. In some embodiments of the invention, the tracrRNA sequence has at least 96% sequence identity compared to any one of SEQ ID NO: 923-1183. In some embodiments of the invention, the tracrRNA sequence has at least 97% sequence identity compared to any one of SEQ ID NO: 923-1183.
  • the tracrRNA sequence has at least 98% sequence identity compared to any one of SEQ ID NO: 923-1183. In some embodiments of the invention, the tracrRNA sequence has 100% sequence identity compared to any one of SEQ ID NO: 923-1183. In some embodiments of the present invention, the tracrRNA sequence is optionally selected from any one of SEQ ID NO:923-1183.
  • the tracrRNA may be complementary to the same repeat sequence.
  • the complementary pairing is a complementary pairing of partial bases.
  • the tracrRNA may interact with the same repeat sequence.
  • the tracrRNA sequence is connected to the same repeat sequence. In a specific embodiment of the present invention, the tracrRNA sequence is connected to the same repeat sequence through a nucleotide sequence. In a specific embodiment of the present invention, the tracrRNA sequence is connected to the same repeat sequence through a nucleotide sequence consisting of 1-10 nucleotides. In a specific embodiment of the present invention, the tracrRNA sequence is connected to the same repeat sequence through a nucleotide sequence consisting of 1-10 nucleotides. The complex sequence is connected by a nucleotide sequence consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.
  • the tracrRNA sequence is connected to the direct repeat sequence by a nucleotide sequence consisting of 4 nucleotides. In a specific embodiment of the present invention, the tracrRNA sequence is connected to the direct repeat sequence by a 5'-GAAA-3' sequence.
  • the tracrRNA sequence is located at the 3' end of the direct repeat sequence.
  • the tracrRNA sequence is located at the 5' end of the direct repeat sequence.
  • the tracrRNA comprises 10-200 nucleotides. In a specific embodiment of the present invention, the tracrRNA comprises 10-190, 10-180, 10-170, 10-160, 10-150, 10-140, 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10- In some embodiments, the present invention relates to a polypeptide having at least one nucleotide sequence and at least one nucleotide sequence. In some embodiments, the present invention relates to a polypeptide having at least one nucleotide sequence and at least one nucleotide sequence.
  • the tracrRNA comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,
  • the tracrRNA sequences corresponding to some Cas proteins are shown in the sequence table.
  • the corresponding gRNA may contain only the guide sequence and the DR sequence, but not the tracrRNA sequence.
  • the gRNA may or may not contain the tracrRNA sequence in addition to the guide sequence and the DR sequence.
  • a technical solution provided by the present invention is: an inactivated variant of the Cas protein, characterized in that the inactivated variant of the Cas protein is a nuclease activity inactivated variant of the Cas protein as described in the present invention.
  • the inactivated variant of the Cas protein is a variant in which the nuclease activity is completely inactivated, that is, a dead Cas protein inactivated variant (dCas protein).
  • the dCas protein can only bind to the target nucleic acid under the mediation of the guide polynucleotide, and has no or almost no function of cutting the target nucleic acid.
  • the target nucleic acid cutting efficiency of the dCas protein is ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, ⁇ 4%, ⁇ 6%, ⁇ 7%, ⁇ 8%, ⁇ 9%, ⁇ 10%, ⁇ 11%, ⁇ 12%, ⁇ 13%, ⁇ 14%, ⁇ 16%, ⁇ 17%, ⁇ 18%, ⁇ 19%, ⁇ 20%, ⁇ 21%, ⁇ 22%, ⁇ 23%, ⁇ 24%, ⁇ 25%, ⁇ 26%, ⁇ 27%, ⁇ 28%, ⁇ 29%, ⁇ 30%, ⁇ 31%, ⁇ 32%, ⁇ 33%, ⁇ 34%, ⁇ 35%, ⁇ 36%, ⁇ 37%, ⁇ 38%, ⁇ 39%, ⁇ 40%, ⁇ 41%, ⁇ 42%, ⁇ 43%, ⁇ 44%, ⁇ 45%, ⁇ 46%, ⁇ 47%, ⁇ 48%, ⁇ 49%, ⁇ 50
  • the inactivated variant of the Cas protein is a variant with partially inactivated nuclease activity.
  • the variant with partially inactivated nuclease activity is a Cas protein nickase (nickase Cas protein, nCas protein), which binds to the target nucleic acid under the mediation of the guide polynucleotide, and then cuts one of the single strands in the double-stranded target nucleic acid without cutting the other single strand.
  • the inactivated variant of the Cas protein is an inactivated Ruvc domain of the Cas protein.
  • the inactivated variant of the Cas protein is an inactivated Ruvc-I, Ruvc-II or Ruvc-III domain of the Cas protein.
  • the inactivated variant of the Cas protein is obtained by introducing an inactivating mutation into the Ruvc-I, Ruvc-II or Ruvc-III domain of the Cas protein.
  • the PAM sequence recognizable by the inactivated variant of the Cas protein is the same as the PAM sequence recognizable by the Cas protein.
  • a technical solution provided by the present invention is: a fusion protein or conjugate, wherein the fusion protein or conjugate comprises the following elements: (1) the Cas protein as described in the present invention, or the inactivated variant of the Cas protein as described in the present invention; and (2) a homologous or heterologous functional domain.
  • a fusion protein which comprises: (1) a Cas protein as described in the present invention, or an inactivated variant of the Cas protein as described in the present invention; and (2) a homologous or heterologous functional domain.
  • a fusion protein which comprises: (1) the Cas protein as described in the present invention; and (2) a homologous or heterologous functional domain.
  • a conjugate which comprises: (1) a Cas protein as described in the present invention, or an inactivated variant of the Cas protein as described in the present invention; and (2) a homologous or heterologous functional domain.
  • a conjugate which comprises: (1) a Cas protein as described in the present invention; and (2) a homologous or heterologous functional domain.
  • the homologous or heterologous functional domains are optionally selected from one or more of the following: subcellular localization signals, DNA binding domains, protease domains, transcription activation domains, transcription repression domains, nuclease domains, deaminase domains, uracil DNA glycosylase domains (UDG), uracil DNA glycosylase inhibitory domains (UGI), methylases, demethylases, transcription release factors, histone acetylase domains, histone deacetylase domains, DNA ligases, affinity tags, reporter tags, affinity domains and reporter domains.
  • subcellular localization signals DNA binding domains, protease domains, transcription activation domains, transcription repression domains, nuclease domains, deaminase domains, uracil DNA glycosylase domains (UDG), uracil DNA glycosylase inhibitory domains (UGI), methylases, demethylases, transcription release factors, histone
  • the subcellular localization signal is selected from: a nuclear localization signal, a nuclear export signal, a mitochondrial localization signal, and a chloroplast localization signal.
  • the fusion protein or conjugate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or more of the homologous or heterologous functional domains; the functional domains are the same or different.
  • the fusion protein or conjugate is arbitrarily linked to 0, 1, 2, 3, 4, 5, 6, 7, 8 or more of the protein domains at the N-terminus and/or C-terminus of the Cas protein.
  • the fusion protein comprises 1, 2, 3, 4 or more nuclear localization signals.
  • the fusion protein can be used to achieve base editing, for example, in combination with a guide polynucleotide to achieve base editing.
  • the fusion protein comprises a nuclear localization signal and a deaminase domain.
  • the fusion protein comprises a nuclear localization signal and a cytidine deaminase domain.
  • the fusion protein can be used to achieve C ⁇ T base editing.
  • the fusion protein comprises a nuclear localization signal and an adenosine deaminase domain.
  • the fusion protein can be used to achieve A ⁇ G base editing.
  • the fusion protein comprises a nuclear localization signal, a cytidine deaminase domain, and an adenosine deaminase domain. In a specific embodiment of the present invention, the fusion protein comprises 1, 2 or 3 nuclear localization signals, and a deaminase domain. In a specific embodiment of the present invention, the fusion protein comprises a UGI domain. In a specific embodiment of the present invention, the fusion protein comprises 1, 2 or 3 nuclear localization signals, a deaminase domain, and 1 or 2 UGI domains.
  • the fusion protein can be used to achieve transcriptional activation of a specific target gene, for example, in combination with a guide polynucleotide to achieve transcriptional activation of a specific target gene.
  • the fusion protein comprises a nuclear localization signal and a transcriptional activation domain.
  • the fusion protein can be used to achieve transcriptional inhibition of a specific target gene, for example, in combination with a guide polynucleotide to achieve transcriptional inhibition of a specific target gene.
  • the fusion protein comprises a nuclear localization signal and a transcriptional inhibition domain.
  • the fusion protein can be used to achieve methylation of a specific target sequence, for example, in combination with a guide polynucleotide to achieve methylation of a specific target sequence.
  • the fusion protein contains a nuclear localization signal and a DNA methylation domain.
  • the fusion protein can be used to achieve demethylation of a specific target sequence, for example, in combination with a guide polynucleotide to achieve demethylation of a specific target sequence.
  • the fusion protein comprises a nuclear localization signal and a DNA demethylation domain.
  • the nuclease domain comprises a polypeptide having ssDNA cleavage activity and/or a polypeptide having dsDNA cleavage activity.
  • the nuclease domain comprises a polypeptide having ssDNA cleavage activity.
  • the nuclease domain comprises a polypeptide having dsDNA cleavage activity.
  • the Cas protein or inactivated variant is directly or indirectly connected to the homologous or heterologous functional domain.
  • the direct connection is covalent connection
  • the indirect connection is connection via an amino acid linker or a non-amino acid linker.
  • the homologous or heterologous functional domain is fused or conjugated at the N-terminus, C-terminus or inside the Cas protein or inactivated variant.
  • the fusion protein refers to the element (1) and the element (2) being connected via a peptide segment, or being directly connected; the conjugate refers to the element (1) and the element (2) being connected via a non-peptide chemical bond.
  • the PAM sequence recognizable by the fusion protein or conjugate is the same as the PAM sequence recognizable by the Cas protein.
  • a technical solution provided by the present invention is: an isolated nucleic acid, which encodes the Cas protein as described in the present invention, the inactivated variant of the Cas protein as described in the present invention, or the fusion protein or conjugate as described in the present invention.
  • the nucleic acid encodes a Cas protein as described in the invention or a fusion protein as described in the invention.
  • the nucleic acid is codon optimized for expression in a cell.
  • the nucleic acid is codon optimized for expression in a eukaryote, a mammal such as a human or non-human mammal, a plant, an insect, a bird, a reptile, a rodent (e.g., a mouse, a rat), a fish, a worm/nematode or a yeast.
  • a mammal such as a human or non-human mammal
  • a plant an insect, a bird, a reptile, a rodent (e.g., a mouse, a rat), a fish, a worm/nematode or a yeast.
  • a technical solution provided by the present invention is: a CRISPR-Cas protein system, the CRISPR-Cas protein system comprising:
  • the Cas protein as described in the present invention the inactivated variant of the Cas protein as described in the present invention, the fusion protein or conjugate as described in the present invention, or the nucleic acid as described in the present invention;
  • b a guide polynucleotide, or a polynucleotide sequence encoding the guide polynucleotide
  • the Cas protein, the inactivated variant of the Cas protein, the fusion protein or the conjugate forms a complex with the guide polynucleotide;
  • the guide polynucleotide comprises a guide sequence, and the guide sequence is engineered to guide the sequence-specific binding of the complex to the target nucleic acid.
  • the guide polynucleotide comprises a direct repeat sequence linked to a guide sequence.
  • the homologous repeated sequence has at least 50% identity with any one of SEQ ID NO:462-922.
  • the guide polynucleotide comprises a direct repeat sequence connected to the guide sequence.
  • the direct repeat sequence has at least 50% identity compared to any one of SEQ ID NO: 462-922.
  • the direct repeat sequence has 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity with the sequence shown in any one of SEQ ID NO: 462-922.
  • the homeotropic repeat sequence comprises or is a sequence shown in any one of SEQ ID NO:462-922.
  • the guide sequence comprises 15-60 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-50 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-40 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-35 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-30 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-25 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 18-25 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 20-25 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 18-22 nucleotides.
  • the guide sequence comprises 20-22 nucleotides. In specific embodiments of the invention, the guide sequence comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.
  • the guide sequence hybridizes to the target nucleic acid, and the guide sequence is 90%-100% complementary to the target nucleic acid.
  • said guide sequence hybridizes to said target nucleic acid.
  • the guide sequence hybridizes to the target nucleic acid, and the guide sequence is mismatched with the target nucleic acid by no more than one nucleotide.
  • the direct repeat sequence comprises 15-100 nucleotides. In a specific embodiment of the invention, the direct repeat sequence comprises 15-90 nucleotides. In a specific embodiment of the invention, the direct repeat sequence comprises 15-80 nucleotides. In a specific embodiment of the invention, the direct repeat sequence comprises 15-70 nucleotides. In a specific embodiment of the invention, the direct repeat sequence comprises 15-60 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-50 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 15-40 nucleotides. In a specific embodiment of the invention, the guide sequence comprises 20-40 nucleotides.
  • the guide sequence comprises 20-30 nucleotides. In specific embodiments of the invention, the guide sequence comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides.
  • the guide sequence is located at the 3' end of the direct repeat sequence.
  • the guide sequence is located at the 5' end of the direct repeat sequence.
  • the guide polynucleotide further comprises tracrRNA.
  • the tracrRNA sequence is at least 50% identical to any one of SEQ ID NOs: 923-1183. In some embodiments of the invention, the tracrRNA sequence is 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to any one of SEQ ID NOs: 923-1183.
  • the tracrRNA may be complementary to the same repeat sequence.
  • the complementary pairing is a complementary pairing of partial bases.
  • the tracrRNA may interact with the same repeat sequence.
  • the tracrRNA sequence is connected to the same repeat sequence. In a specific embodiment of the present invention, the tracrRNA sequence is connected to the same repeat sequence by a nucleotide sequence. In a specific embodiment of the present invention, the tracrRNA sequence is connected to the same repeat sequence by a nucleotide sequence consisting of 1-10 nucleotides. In a specific embodiment of the present invention, the tracrRNA sequence is connected to the same repeat sequence by a nucleotide sequence consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.
  • the tracrRNA sequence is connected to the same repeat sequence by a nucleotide sequence consisting of 4 nucleotides. In a specific embodiment of the present invention, the tracrRNA sequence is connected to the same repeat sequence by a 5'-GAAA-3' sequence.
  • the tracrRNA sequence is located at the 3' end of the direct repeat sequence.
  • the tracrRNA sequence is located at the 5' end of the direct repeat sequence.
  • the tracrRNA comprises 10-200 nucleotides. In a specific embodiment of the present invention, the tracrRNA comprises 10-190, 10-180, 10-170, 10-160, 10-150, 10-140, 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10- In some embodiments, the present invention relates to a polypeptide having at least one nucleotide sequence and at least one nucleotide sequence. In some embodiments, the present invention relates to a polypeptide having at least one nucleotide sequence and at least one nucleotide sequence.
  • the tracrRNA comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,
  • the guiding polynucleotide is the guiding polynucleotide as described in the present invention.
  • the target nucleic acid is DNA or RNA, preferably dsDNA or ssDNA.
  • the DNA is eukaryotic DNA; preferably, the eukaryotic DNA is non-human mammal DNA, non-human primate DNA, human DNA, plant DNA, insect DNA, bird DNA, reptile DNA, rodent DNA, fish DNA, worm/nematode DNA or yeast DNA.
  • the target nucleic acid is a disease or disorder related gene or a signal transduction biochemical pathway.
  • the target nucleic acid is a reporter gene; for example, the disease or disorder is a blood disease or disorder, an ophthalmic disease or disorder, a nervous system disease or disorder, a respiratory system disease or disorder, a liver disease or disorder, a metabolic system disease or disorder, cancer or an infectious disease.
  • a technical solution provided by the present invention is: a vector system, the vector system comprising one or more recombinant vectors, the recombinant vector comprising the isolated nucleic acid as described in the present invention, or the CRISPR-Cas protein system as described in the present invention.
  • the recombinant vector further comprises a regulatory sequence.
  • the vector system comprises one or more recombinant vectors, which contain a polynucleotide sequence encoding the Cas protein, Cas protein inactivated variant or fusion protein or conjugate of the present invention, and a polynucleotide sequence encoding the guide polynucleotide.
  • the polynucleotide sequence encoding the Cas protein, the inactivated variant of the Cas protein, or the fusion protein or conjugate is operably linked to the regulatory sequence 1.
  • the polynucleotide sequence encoding the guide polynucleotide is operably linked to the regulatory sequence 2.
  • the regulatory sequence 1 and the regulatory sequence 2 are identical or different sequences.
  • the regulatory sequence is optionally selected from: one or more of a promoter, an enhancer, an internal ribosome entry site and a transcription termination signal;
  • the promoter is, for example, a constitutive promoter, an inducible promoter, a broad-spectrum promoter or a tissue-specific promoter, and/or the transcription termination signal is, for example, a polyadenylation signal or a poly-U sequence.
  • the backbone of the recombinant vector is an adeno-associated virus vector, a lentivirus vector, a ribonucleoprotein complex or a virus-like particle.
  • the adeno-associated virus vector is a recombinant adeno-associated virus vector of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13;
  • the lentiviral vector is pseudotyped with an envelope protein; preferably, the isolated nucleic acid is linked to an aptamer sequence;
  • the isolated nucleic acid is linked to a gene encoding a gag protein.
  • the present invention provides a technical solution: a delivery system, the delivery system comprising: (1) a delivery vehicle, and (2) a Cas protein as described in the present invention, a guide polynucleotide as described in the present invention, an inactivated variant of a Cas protein as described in the present invention, a fusion protein or conjugate as described in the present invention, a nucleic acid as described in the present invention, a CRISPR-Cas protein system as described in the present invention, or a vector system as described in the present invention.
  • the delivery vehicle is a virus, a lipid nanoparticle, a nanoparticle, a liposome, an exosome, a microbubble or a gene gun.
  • the delivery vehicle is a lipid nanoparticle, which comprises the guide polynucleotide and mRNA encoding the Cas protein, the inactivated variant of the Cas protein, or the fusion protein or conjugate.
  • a technical solution provided by the present invention is: a cell, which comprises the Cas protein as described in the present invention, the guiding polynucleotide as described in the present invention, the inactivated variant of the Cas protein as described in the present invention, the fusion protein or conjugate as described in the present invention, the nucleic acid as described in the present invention, the CRISPR-Cas protein system as described in the present invention, or the vector system as described in the present invention.
  • the cell is a prokaryotic cell.
  • the cell is a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell.
  • a technical solution provided by the present invention is: a pharmaceutical composition, which comprises the Cas protein as described in the present invention, the guiding polynucleotide as described in the present invention, the inactivated variant of the Cas protein as described in the present invention, the fusion protein or conjugate as described in the present invention, the nucleic acid as described in the present invention, the CRISPR-Cas protein system as described in the present invention, the vector system as described in the present invention, the delivery system as described in the present invention or the cell as described in the present invention.
  • the pharmaceutical composition comprises a pharmaceutically acceptable excipient.
  • a technical solution provided by the present invention is: a kit, comprising the Cas protein as described in the present invention, the guiding polynucleotide as described in the present invention, the inactivated variant of the Cas protein as described in the present invention, the fusion protein or conjugate as described in the present invention, the nucleic acid as described in the present invention, the CRISPR-Cas protein system as described in the present invention, the vector system as described in the present invention, the delivery system as described in the present invention or the cell as described in the present invention.
  • the kit further comprises a cutting buffer.
  • the cutting buffer can be any buffer known in the art that is suitable for Cas protein to cut the target nucleic acid.
  • a technical solution provided by the present invention is: the Cas protein as described in the present invention, the guiding polynucleotide as described in the present invention, the inactivated variant of the Cas protein as described in the present invention, the fusion protein or conjugate as described in the present invention, the nucleic acid as described in the present invention, the CRISPR-Cas protein system as described in the present invention, Use of a carrier system, a delivery system as described in the present invention, a cell as described in the present invention, a pharmaceutical composition as described in the present invention or a kit as described in the present invention in the preparation of an agent or drug for diagnosing, treating and/or preventing a disease or condition associated with a target nucleic acid.
  • the disease or condition is a blood system disease or condition, an ophthalmic disease or condition, a nervous system disease or condition, a respiratory system disease or condition, a liver disease or condition, a metabolic system disease or condition, cancer or an infectious disease; and/or the agent or drug is used to: cut one or more target nucleic acid molecules or make a nick in one or more target nucleic acid molecules, activate or upregulate the expression of one or more target nucleic acid molecules, activate or inhibit the transcription of one or more target nucleic acid molecules, inactivate one or more target nucleic acid molecules, visualize, label or detect one or more target nucleic acid molecules, bind one or more target nucleic acid molecules, transport one or more target nucleic acid molecules, and mask one or more target nucleic acid molecules.
  • a technical solution provided by the present invention is: a method for detecting, binding or cutting a target nucleic acid, the method comprising contacting the target nucleic acid with the Cas protein as described in the present invention, the guiding polynucleotide as described in the present invention, the inactivated variant of the Cas protein as described in the present invention, the fusion protein or conjugate as described in the present invention, the nucleic acid as described in the present invention, the CRISPR-Cas protein system as described in the present invention, the vector system as described in the present invention, the delivery system as described in the present invention, the cell as described in the present invention, the pharmaceutical composition as described in the present invention or the kit as described in the present invention.
  • the method is a method for non-diagnostic and/or therapeutic purposes; and/or the fusion protein or conjugate comprises a detectable label, such as a label detectable by fluorescence, Southern blot or FISH.
  • the method when the method is for cutting a target nucleic acid, the method further comprises using a cutting buffer to perform a cutting reaction.
  • the cutting buffer can be any buffer known in the art that is suitable for Cas protein to cut a target nucleic acid.
  • a technical solution provided by the present invention is: a method for changing a cell state, the method comprising contacting a cell with a Cas protein as described in the present invention, a guiding polynucleotide as described in the present invention, an inactivated variant of a Cas protein as described in the present invention, a fusion protein or conjugate as described in the present invention, a nucleic acid as described in the present invention, a CRISPR-Cas protein system as described in the present invention, a vector system as described in the present invention, a delivery system as described in the present invention, a cell as described in the present invention, a pharmaceutical composition as described in the present invention, or a kit as described in the present invention, thereby changing the cell state.
  • the method results in one or more of the following: increase or decrease in expression of a specific gene, induction of cell senescence in vitro or in vivo, cell cycle arrest in vitro or in vivo, cell growth promotion and/or cell growth inhibition in vitro or in vivo, induction of anergy in vitro or in vivo, induction of cell apoptosis in vitro or in vivo, and induction of necrosis in vitro or in vivo.
  • the method is a method for non-diagnostic and/or therapeutic purposes.
  • a technical solution provided by the present invention is: a method for diagnosing, treating or preventing a disease or condition associated with a target nucleic acid, administering a Cas protein as described in the present invention, a guiding polynucleotide as described in the present invention, an inactivated variant of a Cas protein as described in the present invention, a fusion protein or conjugate as described in the present invention, a nucleic acid as described in the present invention, a CRISPR-Cas protein system as described in the present invention, a vector system as described in the present invention, a delivery system as described in the present invention, a cell as described in the present invention, a pharmaceutical composition as described in the present invention, or a kit as described in the present invention to a sample of a subject in need or to a subject in need.
  • the disease or disorder is a blood system disease or disorder, an ophthalmic disease or disorder, a nervous system disease or disorder, a respiratory system disease or disorder, a liver disease or disorder, a metabolic system disease or disorder, cancer or an infectious disease.
  • a technical solution provided by the present invention is: the Cas protein as described in the present invention, the guiding polynucleotide as described in the present invention, the inactivated variant of the Cas protein as described in the present invention, the fusion protein or conjugate as described in the present invention, the nucleic acid as described in the present invention, the CRISPR-Cas protein system as described in the present invention, the vector system as described in the present invention, the delivery system as described in the present invention, the cell as described in the present invention, the pharmaceutical composition as described in the present invention or the kit as described in the present invention, which is used for diagnosing, treating or preventing diseases or disorders associated with target nucleic acids.
  • the disease or disorder is a blood system disease or disorder, an ophthalmic disease or disorder, a nervous system disease or disorder, a respiratory system disease or disorder, a liver disease or disorder, a metabolic system disease or disorder, cancer or an infectious disease.
  • the reagents and raw materials used in the present invention are commercially available.
  • plurality refers to greater than or equal to two.
  • the letters in the amino acid sequence represent the single-letter abbreviations of amino acids known in the art, such as those described in J.Biol.Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.
  • amino acid difference refers to the difference in amino acid residues at specific sites on the amino acid sequence of a protein, including substitution, addition or reduction.
  • amino acid residues In addition, in order to simplify the expression, the amino acid residue before substitution is retained in front of the site where the amino acid residue is located in the present disclosure, the letter before the site represents the original amino acid residue, and the letter after the site represents the amino acid residue after substitution.
  • S211 represents that the original amino acid residue at the 211 site is S, and when it is replaced by R, it can be expressed as S211R.
  • an amino acid if an amino acid is substituted, it means that it is substituted with another amino acid residue different from the original amino acid residue. If the original amino acid was originally a positively charged amino acid, and it is replaced with a positively charged amino acid, it means that it is replaced with another positively charged amino acid residue different from the original amino acid residue. For example, if the original amino acid residue is R, and it is replaced with a positively charged amino acid, it means that it is replaced with H or K.
  • identity As used herein, the term “identity” (identity or percent identity) is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a certain position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or a certain position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position.
  • the "percent sequence identity” (percent identity) between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared ⁇ 100%.
  • the two sequences have 60% sequence identity.
  • the comparison is made when the two sequences are aligned to produce maximum sequence identity.
  • Such an alignment can be performed using published and commercially available alignment algorithms and programs, such as, but not limited to, Clustal ⁇ , MAFFT, Probcons, T-Coffee, Probalign, BLAST, and general algorithms in the art.
  • a skilled person can reasonably choose to use.
  • a skilled person can determine suitable parameters for comparing sequences, for example, including any algorithm required for achieving a better comparison or optimal comparison over the entire length of the compared sequence, and any algorithm required for achieving a better comparison or optimal comparison over the local portion of the compared sequence.
  • CRISPR-CRISPR-associated (Cas) CRISPR-Cas System
  • CRISPR System CRISPR System
  • a transcription product or other element may include a sequence encoding a Cas effector protein and a guide polynucleotide.
  • Zhang Feng's research group discovered Cas protein a, which was classified as type V in the Class II CRISPR-Cas system. After a detailed study of the V-A subtype (Cas protein a), Zhang Feng's research group reported Cas protein b (C2C1) in 2015. In 2017, Burstein et al. reported Cas protein e (CasX) nuclease. In 2019, Winston X. Yan et al. reported in detail the newly discovered V-type Cas effector proteins Cas protein c, Cas protein h, Cas protein i and Cas protein g through bioinformatics analysis.
  • the Cas protein described herein refers to a protein having an amino acid sequence comprising or being 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity compared to any one of SEQ ID NOs: 1-461.
  • the CRISPR-Cas protein system comprises a fusion protein or conjugate comprising the Cas protein and a protein domain
  • the percentage of sequence identity between the Cas protein portion of the fusion protein or conjugate and the reference sequence is calculated.
  • the CRISPR-Cas protein system comprises a Cas protein having at least 50% sequence identity with any one of SEQ ID NO: 1-461, or a nucleic acid encoding the Cas protein; and a guide polynucleotide or a nucleic acid encoding the guide polynucleotide;
  • the guide polynucleotide comprises a direct repeat sequence connected to a guide sequence, the guide sequence is engineered to hybridize with a target nucleic acid, and the guide polynucleotide is capable of forming a complex with the Cas protein and guiding the complex to bind sequence-specifically to the target nucleic acid.
  • the term "guide polynucleotide” is used interchangeably with “guide RNA” to refer to a molecule in the CRISPR-Cas system that forms a complex with the Cas protein and guides the complex to the target sequence.
  • the nucleotide comprises a backbone sequence connected to a guide sequence, and the guide sequence can hybridize with a target sequence.
  • the backbone sequence generally comprises a direct repeat sequence and sometimes may also comprise a tracrRNA sequence.
  • the guide polynucleotide does not comprise a tracrRNA sequence.
  • the guide polynucleotide comprises a tracrRNA sequence.
  • the guide polynucleotide of the CRISPR-Cas protein system is a guide RNA. In some embodiments, the guide polynucleotide is a chemically modified guide polynucleotide. In some embodiments, the guide polynucleotide comprises at least one chemically modified nucleotide.
  • the guide polynucleotide comprises at least one guide sequence (also called a spacer sequence) connected to at least one direct repeat sequence (DR).
  • the guide sequence is located at the 3' end of the direct repeat sequence. In some embodiments, the guide sequence is located at the 5' end of the direct repeat sequence.
  • the tracrRNA sequence is linked to the direct repeat sequence.
  • the tracrRNA sequence is located at the 5' or 3' end of the direct repeat sequence. In some embodiments, the tracrRNA sequence is located at the 5' end of the direct repeat sequence. In some embodiments, the tracrRNA sequence is located at the 3' end of the direct repeat sequence.
  • the nucleotide sequence of the guide polynucleotide comprises, from 5' to 3', tracrRNA, a direct repeat sequence, and a guide sequence.
  • the nucleotide sequence of the guide polynucleotide comprises, from 5' to 3', tracrRNA, a linker sequence, a direct repeat sequence, and a guide sequence.
  • the nucleotide sequence of the guide polynucleotide comprises, from 5' to 3', tracrRNA, loop sequence, direct repeat sequence, and guide sequence.
  • the structure of the guide polynucleotide is 5'-tracrRNA-loop-direct repeat sequence-guide sequence-3'.
  • the tracrRNA and direct repeat sequences of the guide polynucleotide are linked by a nucleotide sequence.
  • the tracrRNA sequence is connected to the same repeat sequence by a nucleotide sequence consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In a specific embodiment of the present invention, the tracrRNA sequence is connected to the same repeat sequence by a nucleotide sequence consisting of 4 nucleotides. In a specific embodiment of the present invention, the tracrRNA sequence is connected to the same repeat sequence by a 5'-GAAA-3' sequence.
  • the guide sequence has sufficient complementarity to the target nucleic acid sequence to bind to the target nucleic acid sequence.
  • the guide sequence is a nucleic acid that hybridizes and guides the CRISPR-Cas protein complex to sequence-specific binding to the target nucleic acid.
  • the guide sequence has 100% complementarity with the target nucleic acid, but the guide sequence can have less than 100% complementarity with the target nucleic acid, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%.
  • the guide sequence is engineered to hybridize to the target nucleic acid with no more than two nucleotide mismatches. In some embodiments, the guide sequence is engineered to hybridize to the target nucleic acid with no more than one nucleotide mismatches. In some embodiments, the guide sequence is engineered to hybridize to the target nucleic acid with or without mismatches.
  • the CRISPR-Cas protein system comprises at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 different guide polynucleotides.
  • the guide polynucleotides target at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 different target nucleic acid molecules, or target at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 different regions of one or more target nucleic acid molecules.
  • the guide polynucleotide includes a constant direct repeat sequence located upstream of the variable guide sequence.
  • multiple guide polynucleotides are part of an array (which can be part of a vector, such as a viral vector or a plasmid).
  • a guide array including the sequence DR-spacer-DR-spacer-DR-spacer- whil-DR-spacer can include multiple unique unprocessed guide polynucleotides (one for each DR-spacer or spacer-DR sequence).
  • the array is processed by the Cas protein into several separate mature guide polynucleotides. This allows multiplexing, such as delivering multiple guide polynucleotides to a cell or system to target multiple target nucleic acids or multiple regions within a single target nucleic acid.
  • CRISPR complex The ability of a guide polynucleotide to guide a complex (CRISPR complex) to bind sequence-specifically to a target nucleic acid can be assessed by any suitable assay.
  • components of a CRISPR system sufficient to form a complex (CRISPR complex) can be provided to a host cell having a corresponding target nucleic acid molecule, such as by transfection of a vector encoding components of the CRISPR complex, and then preferential cleavage within the target sequence can be assessed.
  • cleavage of a target nucleic acid sequence can be assessed in a test tube by providing a target nucleic acid, components of a CRISPR complex, including a guide polynucleotide to be tested and a control guide polynucleotide different from the test guide polynucleotide, and comparing the ability to bind to the target nucleic acid or the rate of cleavage of the target nucleic acid between the guide polynucleotide to be tested and the control.
  • the ability of a CRISPR complex to cleave a target nucleic acid or a target nucleic acid can also be assessed by the above-described assays.
  • the Cas protein provided herein comprises one or more mutations, such as a single amino acid insertion, a single amino acid deletion, a single amino acid substitution, or a combination thereof, compared to a wild-type Cas protein (SEQ ID NO: 1-461).
  • the Cas protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 3, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 129, 130, 131,
  • One type of modification or mutation includes replacing amino acid residues with similar biochemical properties with amino acids, i.e., conservative substitutions.
  • conservative substitutions typically have little or no effect on the activity of the resulting protein or peptide.
  • conservative substitutions are amino acid substitutions in the Cas protein that do not substantially affect the binding of the Cas protein to the target nucleic acid molecule complementary to the guide sequence of the gRNA molecule, and/or the process of processing the guide array RNA transcript into a gRNA molecule.
  • More substantial changes can be made by using less conservative substitutions, for example, by selecting residues that differ more in maintaining: (a) the structure of the polypeptide backbone in the region where the substitution occurs, for example, as a helical or folded conformation; (b) the charge or hydrophobicity of the region that interacts with the target site; or (c) the bulk of the side chain.
  • substitutions that would generally be expected to produce the greatest changes in polypeptide function are (a) substitutions of a hydrophilic residue (e.g., serine or threonine) with a hydrophobic residue (e.g., (a) between cysteine or proline and any other residue; (b) between a residue with a positively charged side chain (e.g., lysine, arginine, or histidine) and a negatively charged residue (e.g., glutamic acid or aspartic acid); or (c) between a residue with a bulky side chain (e.g., phenylalanine) and a residue without a side chain (e.g., glycine).
  • a hydrophilic residue e.g., serine or threonine
  • a hydrophobic residue e.g., (a) between cysteine or proline and any other residue
  • a residue with a positively charged side chain e.g., lysine, argin
  • the Cas protein may only include a WED-I domain, a Helical-I1 domain, a PI domain, a Helical-I2 domain, a Helical-II domain, a WED-II domain, a Ruvc-I domain, a Helical-III domain, a BH domain, a Ruvc-II domain, a Nuc domain and/or a Ruvc-III domain.
  • the Cas protein described in the present invention in addition to comprising the domain, may also comprise domains of other Cas proteins in the prior art, which are combined together to form a complete structure of the Cas protein to achieve the function of the Cas protein described in the present invention, including but not limited to retaining the ability of the Cas protein to bind to a target nucleic acid molecule complementary to the guide sequence of the guide polynucleotide, and/or retaining the ability to process an RNA transcript comprising a guide sequence into a guide polynucleotide molecule.
  • the Cas protein By making the RuvC domain of the Cas protein inactive through point mutation, the Cas protein will lose its endonuclease activity.
  • the resulting dCas protein (dead Cas protein) can only bind to the target gene under the mediation of the guiding polynucleotide, but does not have the function of cutting DNA.
  • the RuvC domain of the Cas protein can also lose some of its activity through point mutation, forming a Cas protein nickase (nCas protein), which binds to the target gene under the mediation of the guiding polynucleotide and cuts one of the single strands in the double-stranded nucleic acid without cutting the other single strand.
  • nCas protein Cas protein nickase
  • the dCas protein or nCas protein can be fused or conjugated with other domains (including but not limited to deaminase domains, transcription activation domains, transcription repression domains, methylation domains, demethylation domains, histone acetylation domains, and histone deacetylation domains), guided to the target sequence of the target nucleic acid by the guiding polynucleotide, and then the corresponding functions are performed with the help of the other domains; for example, the conversion of base C ⁇ T is achieved by deaminating cytosine bases, the conversion of base A ⁇ G is achieved by deaminating adenine bases, transcription repression is achieved by the transcription repression domain KRAB, and transcription is promoted by the transcription activation domain VP64.
  • other domains including but not limited to deaminase domains, transcription activation domains, transcription repression domains, methylation domains, demethylation domains, histone acetylation domains
  • the Cas protein or inactive variant of the Cas protein is covalently linked or fused to a homologous or heterologous protein domain.
  • the protein domain is selected from one or more of the following: a subcellular localization signal, a DNA binding domain, a protease domain, a transcriptional activation domain, a transcriptional repression domain, a nuclease domain, a deaminase domain, Uracil DNA glycosylase domain (UDG), uracil DNA glycosylase inhibitory domain (UGI), methylase, demethylase, transcription release factor, histone acetylase domain, histone deacetylase domain, DNA ligase, epitope tag and reporter domain.
  • the deaminase domain is selected from: APOBEC1, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, activation-induced cytidine deaminase (AID), CDA from lamprey, a mutant of adenosine deaminase engineered to act on DNA (TadA).
  • the transcriptional activation domain is selected from: P65, VPR, VP16, VP64, VTR1, VTR2, VTR3, p65, MyoD1, HSF1, RTA, SET7/9 and histone acetyltransferase.
  • the transcriptional activation domain is optionally selected from: sequence ETFSDLWKL from p53TAD1, sequence DDIEQWFTE from p53TAD2, sequence SDIMDFVLK from MLL, sequence DLLDFSMMF from E2A, sequence ETLDFSLVT from Rtg3, sequence RKILNDLSS from CREB, sequence EAILAELKK from CREBaB6, sequence DDVVQYLNS from Gli3, sequence DDVYNYLFD from Gal4, sequence DLFDYDFLV from Oaf1, sequence DFFDYDLLF from Pip2, sequence EDLYSILWS from Pdr1, sequence TDLYHTLWN from Pdr3.
  • the transcriptional repression domain is optionally selected from: KOX1, KAP-1, MAD, FKHR, EGR-1, ERD, SID, a tandem of SID (e.g., SID4X), TIEG, v-ERB-A, MBD2, MBD3, TRa, histone methyltransferase, histone deacetylase (HDAC), a nuclear hormone receptor (e.g., an estrogen receptor or a thyroid hormone receptor), a DNMT family member (e.g., DNMT1, DNMT3A, DNMT3B), the KRAB domain of MeCP2, ROM2, and AtHD2A.
  • SID4X tandem of SID
  • TIEG e.g., v-ERB-A, MBD2, MBD3, TRa
  • HDAC histone methyltransferase
  • HDAC histone deacetylase
  • DNMT family member e.g., DNMT1, DNMT3A, DNMT3B
  • the transcriptional repression domain is a KRAB domain from a KOX1 protein.
  • the nuclease domain is selected from FokI, a polypeptide having ssDNA cleavage activity, and a polypeptide having dsDNA cleavage activity.
  • the methylase domain is selected from DNA methylases, including but not limited to DNMT1, DNMT3a, DNMT3b.
  • the demethylase is selected from TET1CD, TET1, ROS1, DME, DML2, and DML3.
  • Methylation and demethylation are recognized in the art as important means of epigenetic gene regulation.
  • the homologous or heterologous protein domain is a sequence tag useful for dissolution, purification or detection of the fusion protein or conjugate.
  • Suitable protein tag sequences are provided herein, including but not limited to biotin carboxylase carrier protein (BCCP) tags, myc tags, calmodulin tags, FLAG tags, hemagglutinin (HA) tag, polyhistidine tag (also known as His tag), maltose binding protein (MBP) tag, nus tag, glutathione-S-transferase (GST) tag, green fluorescent protein (GFP) tag, thioredoxin tag, S-tag, Softag (e.g., Softag 1, Softag 3), strep-tag, biotin ligase tag, FlAsH tag, V5 tag and SBP tag. Additional suitable sequences will be apparent to those of ordinary skill in the art.
  • BCCP biotin carboxylase carrier protein
  • myc tags myc tags
  • calmodulin tags FLAG tags
  • the Cas protein is fused to at least one homologous or heterologous subcellular localization signal. In some embodiments, the Cas protein is fused to at least one homologous or heterologous subcellular localization signal.
  • Exemplary subcellular localization signals include organelle localization signals, such as nuclear localization signals (NLS), nuclear export signals (NES) or mitochondrial localization signals.
  • Non-limiting examples of NLSs include NLS sequences derived from: the NLS of the SV40 virus large T antigen, which has the amino acid sequence PKKKRKV; the NLS from nucleoplasmic protein (e.g., sequence KRPAATKKAGQAKKKK); c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; hRNPA1M9NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV from the IBB domain; the sequence VSRKRPRP and PPKKARED of myoma T protein; the sequence PQPKKKPL of human p53; the sequence SALIKKKKKMAP of mouse c-ablIV; the sequence DRLRR and PKQKKRK of influenza virus NS1; the sequence RKLKKKIKKL of hepatitis virus delta anti
  • the nuclear localization sequence has sufficient strength to achieve driving the fusion protein or conjugate of the present invention to accumulate in a detectable amount in the nucleus of a eukaryotic cell.
  • the intensity of nuclear localization activity can be derived from the number of NLSs, one or more specific NLSs used, or a combination of these factors.
  • Detection of accumulation in the nucleus can be performed by any suitable technique.
  • a detectable marker can be fused to the Cas protein so that the position in the cell can be visualized, such as combined with a means for detecting the position of the nucleus (e.g., a dye specific to the nucleus, such as DAPI).
  • the nucleus can also be separated from the cell, and then its contents can be analyzed by any suitable method for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus can also be determined indirectly in the following ways: such as by measuring the effect of nucleic acid targeting complex formation (e.g., measuring DNA or RNA cutting or mutation at the target sequence, or measuring gene expression activity that changes due to the influence of DNA or RNA targeting complex formation and/or DNA or RNA targeting Cas protein activity), Comparisons were made to controls that were not exposed to the nucleic acid-targeting Cas protein or nucleic acid-targeting complex, or that were exposed to a nucleic acid-targeting Cas protein lacking one or more NLSs.
  • any suitable method for detecting protein such as immunohistochemistry, Western blot, or enzyme activity assay.
  • Accumulation in the nucleus can also be determined indirectly in the following ways: such as by measuring the effect of nucleic acid targeting complex formation (e.g.,
  • Another aspect of the present disclosure relates to a vector system comprising the CRISPR-Cas protein system described herein, the vector system comprising one or more recombinant vectors comprising a polynucleotide sequence encoding the Cas protein and a polynucleotide sequence encoding the guide polynucleotide.
  • the vector system comprises at least one plasmid or viral recombinant vector (e.g., retrovirus, lentivirus, adenovirus, adeno-associated virus, or herpes simplex virus).
  • the polynucleotide sequence encoding the Cas protein and the polynucleotide sequence encoding the guide polynucleotide are located on the same recombinant vector.
  • the polynucleotide sequence encoding the Cas protein and the polynucleotide sequence encoding the guide polynucleotide are located on multiple recombinant vectors.
  • the polynucleotide sequence encoding the Cas protein and/or the polynucleotide sequence encoding the guide polynucleotide is operably connected to a regulatory sequence (also referred to as a regulatory element).
  • the regulatory element includes a promoter, an enhancer, an internal ribosome entry site (IRES) and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • Regulatory elements include regulatory elements that constitutively express nucleotide sequences in many types of host cells, and regulatory elements that express nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences).
  • Tissue-specific promoters can be directly expressed primarily in desired tissues of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements can also direct expression in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell type-specific.
  • the regulatory element is an enhancer element, such as WPRE, CMV enhancer, R-U5 segment in the LTR of HTLV-1, SV40 enhancer, or the intronic sequence between exons 2 and 3 of rabbit ⁇ -globin.
  • the recombinant vector comprises a pol III promoter (e.g., U6 and H1 promoters), a pol II promoter (e.g., retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter (optionally with CMV enhancer), SV40 promoter, dihydrofolate reductase promoter, ⁇ -actin promoter, phosphoglycerol kinase (PGK) promoter, or EF1 ⁇ promoter), or a pol III promoter and a pol II promoter.
  • a pol III promoter e.g., U6 and H1 promoters
  • a pol II promoter e.g., retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter (optionally with CMV enhancer), SV40 promoter
  • the promoter is a constitutive promoter, which is continuously active and is not regulated by external signals or molecules. Suitable constitutive promoters include, but are not limited to, CMV, RSV, SV40, EF1 ⁇ , CAG and ⁇ -actin promoter. In some embodiments, the promoter is an inducible promoter regulated by an external signal or molecule (eg, a transcription factor).
  • the promoter is a tissue-specific promoter, which can be used to drive tissue-specific expression of Cas proteins.
  • Suitable muscle-specific promoters include, but are not limited to, CK8, MHCK7, myoglobin promoter (Mb), desmin promoter, muscle creatine kinase promoter (MCK) and variants thereof, and SPc5-12 synthetic promoters.
  • Suitable immune cell-specific promoters include, but are not limited to, B29 promoter (B cells), CD14 promoter (monocytes), CD43 promoter (leukocytes and platelets), CD68 (macrophages), and SV40/CD43 promoter (leukocytes and platelets).
  • Suitable blood cell-specific promoters include, but are not limited to, CD43 promoter (leukocytes and platelets), CD45 promoter (hematopoietic cells), INF- ⁇ (hematopoietic cells), WASP promoter (hematopoietic cells), SV40/CD43 promoter (leukocytes and platelets), and SV40/CD45 promoter (hematopoietic cells).
  • Suitable pancreas-specific promoters include, but are not limited to, elastase-1 promoter.
  • Suitable endothelial cell-specific promoters include, but are not limited to, Fit-1 promoter and ICAM-2 promoter.
  • Suitable neuronal tissue/cell-specific promoters include, but are not limited to, GFAP promoter (astroglial cells), SYN1 promoter (neurons) and NSE/RU5' (mature neurons).
  • Suitable kidney-specific promoters include, but are not limited to, NphsI promoter (podocytes).
  • Suitable bone-specific promoters include, but are not limited to, OG-2 promoter (osteoblasts, odontoblasts).
  • Suitable lung-specific promoters include, but are not limited to, SP-B promoter (lung).
  • Suitable liver-specific promoters include, but are not limited to, SV40/Alb promoter.
  • Suitable heart-specific promoters include, but are not limited to, ⁇ -MHC.

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Abstract

Une protéine Cas et son utilisation sont divulguées dans la présente invention. La séquence d'acides aminés de la protéine Cas comprend ou est constituée d'une séquence d'acides aminés ayant au moins 50 % d'identité avec l'une quelconque des SEQ ID NO : 1-461. Un polynucléotide guide, un variant d'inactivation de protéine Cas, une protéine de fusion ou un conjugué comprenant la protéine Cas, un acide nucléique isolé, un système CRISPR-protéine Cas, un système de vecteur, un système de distribution, une cellule, une composition pharmaceutique et un kit, et leurs utilisations sont également divulgués dans la présente invention.
PCT/CN2023/142927 2023-12-28 2023-12-28 Protéine cas et son utilisation Pending WO2024078645A2 (fr)

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CN119804865A (zh) * 2024-12-31 2025-04-11 郑州大学 细胞外囊泡蛋白质作为诊断腹主动脉瘤的生物标志物及应用
WO2025242229A1 (fr) * 2024-05-24 2025-11-27 广州瑞风生物科技有限公司 Protéine cas12 et son utilisation

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JP2022546701A (ja) * 2019-08-27 2022-11-07 アーバー バイオテクノロジーズ， インコーポレイテッド 新規ｃｒｉｓｐｒ ｄｎａターゲティング酵素及びシステム
CN116083398B (zh) * 2021-11-05 2024-01-05 广州瑞风生物科技有限公司 分离的Cas13蛋白及其应用
CA3243006A1 (fr) * 2021-12-21 2025-02-27 Alia Therapeutics Srl Proteines cas de type ii et leurs applications
CN114934031B (zh) * 2022-05-25 2023-08-01 广州瑞风生物科技有限公司 新型Cas效应蛋白、基因编辑系统及用途
CN116144629B (zh) * 2022-09-16 2025-08-05 复旦大学 Cas9蛋白、含有Cas9蛋白的基因编辑系统及应用
CN117165557B (zh) * 2023-08-02 2026-04-21 尧唐(上海)生物科技有限公司 Cas蛋白、其相应的基因编辑系统及应用
CN117230043B (zh) * 2023-11-14 2024-04-12 广州瑞风生物科技有限公司 Cas13蛋白及其应用

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Publication number Priority date Publication date Assignee Title
WO2025242229A1 (fr) * 2024-05-24 2025-11-27 广州瑞风生物科技有限公司 Protéine cas12 et son utilisation
CN119804865A (zh) * 2024-12-31 2025-04-11 郑州大学 细胞外囊泡蛋白质作为诊断腹主动脉瘤的生物标志物及应用

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