WO2023208256A1 - 经分离的Cas13蛋白、基于它的基因编辑系统及其用途 - Google Patents

经分离的Cas13蛋白、基于它的基因编辑系统及其用途 Download PDF

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WO2023208256A1
WO2023208256A1 PCT/CN2023/101348 CN2023101348W WO2023208256A1 WO 2023208256 A1 WO2023208256 A1 WO 2023208256A1 CN 2023101348 W CN2023101348 W CN 2023101348W WO 2023208256 A1 WO2023208256 A1 WO 2023208256A1
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cas13
protein
nuclease
sequence
domain
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French (fr)
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李伟
周琪
胡艳萍
陈阳灿
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Institute of Zoology of CAS
Institute for Stem Cell and Regeneration of CAS
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Institute of Zoology of CAS
Institute for Stem Cell and Regeneration of CAS
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Priority to EP23795688.3A priority patent/EP4516910A4/en
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    • C12Y305/04005—Cytidine deaminase (3.5.4.5)

Definitions

  • the present application is in the field of biotechnology and specifically relates to isolated Cas13 protein, gene editing systems based on it and methods of using them for RNA level gene editing.
  • Gene editing technology makes it possible to modify DNA sequence anchor points, such as the first generation of gene editing tools zinc finger nucleases (ZFNs), and the second generation of gene editing tools similar to small nucleases that activate transcription ( transcription activator-like effector nucleases (TALENs), type II and type V clustered regularly interspaced short palindromic repeats (CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR- associated proteins) can be used to target the genome, but these gene editing systems can only target the genome and foreign DNA, but cannot target RNA in the body.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • CRISPR- associated proteins can be used to target the genome, but these gene editing systems can only target the genome and foreign DNA, but cannot target RNA in the body.
  • Type VI CRISPR/Cas13 system is the natural immune system from archaea and bacteria. Different from previous gene editing tools, it uses the principle of complementary nucleic acid base pairing to identify target RNA sequences and guide Cas effector proteins for targeted cleavage. It has strong applicability, simple design, and high efficiency.
  • type VI-D CRISPR/Cas13 gene editing system is the most widely used type VI CRISPR/Cas system.
  • This system can perform targeted editing of single-stranded RNA in prokaryotes by recognizing and cutting the Protospacer flanking site (PFS) sequence on both sides of the targeted polynucleotide.
  • PFS Protospacer flanking site
  • the type VI-D system has no PFS sequence for targeted editing of RNA.
  • the type VI-D system has a smaller protein volume and has better prospects in gene therapy based on adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • AAV adeno-associated virus
  • Isolated Cas13 nuclease protein the amino acid sequence of the Cas13 protein includes:
  • An engineered Cas13 nuclease effector protein containing:
  • effector protein according to item 3 or 4 which further comprises a functional domain fused to the Cas13 nuclease protein.
  • the functional domain is selected from one or more of the following: translation initiation domain, translation repression domain, transactivation domain, epigenetic modification domain, nuclear Base editing domain, reverse transcriptase domain, reporter domain and nuclease domain.
  • nucleobase editing domain is adenosine deaminase, cytidine deaminase or a catalytic domain fusion thereof.
  • a vector comprising the polynucleotide as described in item 8, preferably the vector is a plasmid or lentivirus.
  • An engineered CRISPR-Cas13 gene editing system containing:
  • crRNA which includes a spacer sequence complementary to the target sequence.
  • the crRNA also includes: The sequences shown in SEQ ID NO. 29 to 56 have direct repeat (DR) sequences with at least 80% identity;
  • the engineered Cas13 nuclease effector protein and the crRNA can form a CRISPR complex that specifically binds to a target nucleic acid containing the target sequence and induces modification of the target nucleic acid.
  • a kit comprising the engineered CRISPR-Cas13 gene editing system as described in item 10.
  • the engineered CRISPR-Cas13 nuclease effector protein according to any one of items 3 to 7 and the engineered CRISPR-Cas13 gene editing system according to item 10 are used to prepare and treat diseases related to nucleic acid mutations in individual cells. or use in medicines for medical conditions.
  • a method for modifying a target nucleic acid contained in a cell comprising contacting the cell with the engineered CRISPR-Cas13 nuclease protein effector protein described in any one of items 3-7, and the engineered CRISPR-Cas13 nuclease protein effector protein of item 10.
  • the Cas13 gene editing system contacts to achieve the modification of the target nucleic acid in the cell.
  • This application also provides:
  • Isolated Cas13 nuclease protein the amino acid sequence of the Cas13 nuclease protein is:
  • Cas13 nuclease protein as described in item 1, wherein the Cas13 nuclease protein is Cas13bt1, Cas13bt2, Cas13g, Cas13h, Cas13i, Cas13j or Cas13k protein, preferably Cas13g3.
  • An engineered Cas13 nuclease effector protein comprising a Cas13 nuclease protein, the Cas13 nuclease protein comprising:
  • effector protein according to item 3 or 4 which further comprises a functional domain fused to the Cas13 nuclease protein.
  • the functional domain is selected from one or more of the following: translation initiation domain, translation repression domain, transactivation domain, epigenetic modification domain, Nucleobase editing domain, reverse transcriptase domain, reporter domain and nuclease domain.
  • nucleobase editing domain is adenosine deaminase, cytidine deaminase or their catalytic domain.
  • a vector comprising the polynucleotide as described in item 8, wherein the vector is a plasmid or a virus, and the virus is preferably a lentivirus.
  • An engineered CRISPR-Cas13 gene editing system containing:
  • crRNA comprising a spacer sequence complementary to a target sequence in a target nucleic acid
  • the engineered Cas13 nuclease effector protein and the crRNA can form a CRISPR complex that specifically binds to a target nucleic acid containing the target sequence and induces modification of the target nucleic acid.
  • DR direct repeat
  • a kit comprising the engineered CRISPR-Cas13 gene editing system as described in item 10 or 11.
  • Cas13 nuclease protein as described in item 1 or 2 engineered CRISPR-Cas13 nuclease effector protein as described in any one of items 3-7, engineered CRISPR as described in item 10 or 11 - Use of the Cas13 gene editing system in the preparation of a medicament for the treatment of a disease or condition associated with nucleic acid mutations in cells of an individual.
  • a method for modifying a cell containing a target nucleic acid comprising contacting the cell with a Cas13 nuclease protein as described in item 1 or 2, or an engineered Cas13 nuclease protein as described in any one of items 3-7.
  • the protein is contacted with the engineered CRISPR-Cas13 gene editing system as described in Item 10 or 11, thereby achieving modification of the target nucleic acid in the cell.
  • Cas13g-k The new Cas13 subtype is named Cas13g-k; the Cas13g protein is approximately 800 amino acids in size, and the Cas13h-k protein is approximately 1100 amino acids in size.
  • Cas13g3 protein can be engineered for programmable RNA editing.
  • Cas13g3 has a volume of 767aa and is considered to be the most efficient small Cas13 protein currently and can be used as a promising mammalian RNA editing tool.
  • Figure 1 shows the flow chart of a total of 75 Cas13 proteins in 5 new subtypes identified through analysis of metagenomic data.
  • Figure 2 shows a schematic diagram of the protein homology between the 7 isoforms and the 4 old isoforms and the average protein size of each isoform.
  • Figure 3 shows another schematic diagram of the protein homology between the 7 isoforms and the 4 old isoforms.
  • Figure 4 shows a schematic diagram of the spacer-directed repeat sequence of the Loci site of Cas13bt1-Cas13k.
  • Figure 5 shows a schematic diagram of the HEPN domain at the C-terminus and N-terminus of Cas13bt1-Cas13k.
  • Figure 6 shows the ratio of RxxxxH motifs of Cas13bt1-Cas13k.
  • Figure 7 is a schematic diagram showing the conservation of the RxxxxH motif sequence of the HEPN domain at the C-terminus and N-terminus of Cas13bt1-Cas13k.
  • Figure 8 shows a schematic diagram of the predicted structure of the DR sequence of Cas13bt1-Cas13k.
  • Figure 9 shows the distribution ratio of Cas13a-Cas13k in microorganisms in various environments.
  • Figure 10 shows the existing ratio of Cas13a-Cas13k in nature.
  • Figures 11A and 11B are schematic diagrams of the experimental process for analyzing the RNA cleavage activity of Cas13 protein in HEK293T cells.
  • Figure 12 is a comparison of the efficiency of knocking down mCherry mRNA using 3'-DR or 5'-DR-crRNA.
  • Figures 13A and 13B are for knocking down mCherry mRNA to screen Cas13 proteins with high editing efficiency. Result graph.
  • Figures 14A and 14B are real-time quantitative reverse transcription PCR (RT-qPCR) results of novel Cas13-mediated ANXA4 mRNA knockdown.
  • Figure 15 shows the quantitative RT-qPCR results of endogenous mRNA knockdown mediated by RfxCas13d, Cas13X1, Cas13bt1-11 and Cas13g3.
  • Figure 16 shows the RT-qPCR results of novel Cas13d-mediated knockdown of ANXA4 mRNA.
  • Figure 17 shows the RNA-seq analysis results of ANXA4 mRNA knockdown mediated by RfxCas13d, Cas13X1, Cas13bt1-11 and Cas13g3.
  • Figures 18A and 18B show the trans activity assessment of RfxCas13d, Cas13X1, Cas13bt1-11 and Cas13g3.
  • Figures 19A and 19B show analysis of optimal spacer length for the Cas13g3 system.
  • Figure 20 shows Cas13g3 system PFS preference analysis.
  • Figure 21 shows the effect of NLS on the interference activity of the Cas13g3 system.
  • Figure 22 shows a comparison of the RNA interference capabilities of the Cas13g3 system and other Cas13 systems.
  • substantially free with respect to a particular component is used herein to mean that the particular component is not purposefully formulated into the composition and/or is present only as a contaminant or in trace amounts. Therefore, the total amount of a particular component resulting from any accidental contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the specific component is present in an amount undetectable by standard analytical methods.
  • the present application provides an isolated Cas13 nuclease protein.
  • an isolated Cas13 nuclease protein is provided, and the amino acid sequence of the Cas13 protein includes: a) the amino acid sequence shown in any one of SEQ ID NO. 1 to 28; or, b) An amino acid sequence that has more than 80% sequence identity with any one of SEQ ID NO. 1 to 28 and has RNA cleavage activity.
  • a Cas13 nuclease protein is provided, wherein the Cas13 protein is Cas13bt1, Cas13bt2, Cas13g, Cas13h, Cas13i, Cas13j or Cas13k protein.
  • Cas13a and C2c2 are used interchangeably. It was originally discovered by researchers at the Broad Institute. In 2015, Shmakov and colleagues used Cas1 as a "bait" to identify CRISPR-associated proteins in bacterial genomes. Through this analysis, they identified 53 candidate genes, divided into three major categories: C2c1, C2c2 and C2c3. C2c1 and C2c3 are somewhat similar to Cpf1, except that they require both tracrRNA and crRNA to cleave DNA targets, whereas Cpf1 only requires crRNA. The difference between C2c2 (that is, Cas13a) and Cas9 is that Cas13a binds and cuts RNA, while Cas9 cuts DNA.
  • Cas9 utilizes tracrRNA and crRNA to bind and cleave DNA targets.
  • Cas13a only requires a 24-base crRNA, which interacts with the Cas13a molecule through a uracil-rich stem-loop structure and promotes target cleavage through a series of conformational changes of Cas13a.
  • Cas13a can also tolerate a single mismatch between crRNA and the target sequence, but if there are two mismatches, the cutting efficiency is greatly reduced.
  • Its PFS sequence (equivalent to PAM sequence) is located at the 3' end of the spacer region and consists of A, U or C bases.
  • Cas13a Another special feature of Cas13a is that once Cas13a recognizes and cleaves the RNA target specified by the crRNA sequence, it enters an enzymatic "activation" state. It will bind and cleave other RNAs regardless of whether they are homologous to the crRNA or whether a PFS is present; this is completely different from Cas9.
  • tracrRNA is not required, only crRNA is required; and crRNA consists of a DR sequence (that is, a direct repeat sequence) and a spacer sequence (a spacer sequence, that is, a nucleotide sequence that is complementary to the target sequence).
  • the present application provides an engineered Cas13 nuclease effector protein
  • an engineered Cas13 nuclease effector protein comprising: a) an amino acid sequence as shown in any one of SEQ ID NO. 1 to 28; or, b) and SEQ ID NO. .An amino acid sequence having at least 80% sequence identity and having RNA cleavage activity in any one of 1 to 28.
  • the nuclease effector protein has a ratio of more than 80%, more than 81%, more than 82%, more than 83%, more than 84%, more than 85%, more than 86%, or more than any one of SEQ ID NO. 1 to 28.
  • an engineered Cas13 nuclease effector protein is provided.
  • the Cas13 nuclease effector protein loses its catalytic activity through amino acid mutation.
  • the Cas13 nuclease protein has a HEPN structure at its C-terminus and N-terminus.
  • the domain (RxxxxH motif) is mutated to form the dCas13 protein.
  • HEPN refers to the two domains contained in Cas13a, which act on Cas13 equivalent to the HNH and RuvC domains on Cas9, to cleave target nucleic acids.
  • the HEPN domain is required for Cas13a to cleave RNA targets.
  • the RxxxxH motifs of the HEPN ribonuclease domain are located at the N-terminus and C-terminus of the Cas13 protein respectively.
  • mutations of key residues in the Cas13a molecule can form Cas13a with missing nuclease activity (dCas13a), which can bind to RNA targets but cannot cleave.
  • the RxxxxH motif is 6 consecutive amino acids, the first amino acid is arginine R, the last one is histidine H, and there can be any amino acid in the middle.
  • the RxxxxH motif is the HEPN motif and has ribonuclease function.
  • an engineered Cas13 nuclease effector protein which further comprises a functional domain fused to the Cas13 nuclease protein.
  • the functional domain is selected from one or more of the following: translation initiation domain, translation repression domain, transactivation domain, epigenetic modification domain, nucleobase editing domain, reverse transcription Enzyme domain, reporter domain and nuclease domain; wherein the nucleobase editing domain is adenosine deaminase, cytidine deaminases or their catalytic domain fusions.
  • the application relates to a polynucleotide.
  • a polynucleotide encoding the aforementioned Cas13 nuclease protein or the aforementioned engineered effector protein is provided.
  • the present application relates to a polynucleotide vector.
  • a polynucleotide vector is provided, preferably the vector is a plasmid or lentivirus.
  • the present application relates to a gene editing system.
  • an engineered CRISPR-Cas13 gene editing system comprising: (a) the aforementioned engineered Cas13 nuclease effector protein or nucleic acid encoding the effector protein; and (b) crRNA, It contains a spacer sequence complementary to the target sequence.
  • the crRNA also includes the sequence shown in SEQ ID NO. 29 to 56 respectively.
  • a direct repeat (DR) sequence with at least 80% sequence identity; wherein the engineered Cas13 nuclease effector protein and the crRNA are capable of forming a CRISPR complex that specifically binds to the target sequence.
  • the direct repeat (DR) sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, At least 87%, at least 88%, at least 89%, 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.
  • the present application relates to a kit.
  • kits comprising the aforementioned engineered CRISPR-Cas13 gene editing system is provided.
  • the application relates to the use of engineered CRISPR-Cas13 nuclease effector proteins.
  • the use of the aforementioned engineered CRISPR-Cas13 nuclease effector protein and the aforementioned engineered CRISPR-Cas13 gene editing system in preparing drugs for treating diseases or conditions related to nucleic acid mutations in individual cells is provided. use in.
  • the application relates to a method of modifying a target nucleic acid contained in a cell.
  • a method for modifying a cell containing a target nucleic acid comprising contacting the cell with the aforementioned engineered CRISPR-Cas13 nuclease protein effector protein and the aforementioned engineered CRISPR-Cas13 gene editing system. Contact, thereby achieving modification of the target nucleic acid in the cell.
  • the present application relates to a use for treating a disease or disorder associated with nucleic acid mutations in an individual.
  • the aforementioned engineered CRISPR-Cas13 nuclease effector protein or the aforementioned engineered CRISPR-Cas13 gene editing system is provided for preparation and use to treat diseases or conditions related to nucleic acid mutations in individuals.
  • the purpose of the drug is provided.
  • Phylogenetic analysis and classification of candidate proteins were performed.
  • the 2253 candidate proteins were first clustered through preliminary clustering using the cluster--min-seq-id 0.5-c 0.7 parameter of the MMseqs software. Then each cluster was clustered through the mmseqs software. The cluster--min-seq-id 0.9-c 0.8 parameter removes redundant proteins.
  • Each cluster was then subjected to multiple sequence alignment through MAFFT software. The alignment results were used to construct a hidden Markov model using hhmake of HH-suite3 software. Then the hidden Markov model files were compared with each other using hhalign of HH-suite3 software. right. Alignment scores between proteins were used to calculate evolutionary distance.
  • sij represents the alignment score between i protein and j protein
  • sji represents the alignment score between j protein and i protein
  • Sij represents the average alignment score between i protein and j protein
  • dij represents the evolutionary distance between i protein and j protein.
  • Evolutionary distance results were clustered using the unweighted group average method (UPGMA). Finally, many known Cas13a-d homologs and 7 different clades were identified.
  • the remaining five branches are new subtypes, which we named Cas13g-k.
  • the size of Cas13g protein is about 800 amino acids, and the size of Cas13h-k protein is about 1100 amino acids ( Figures 1 and 2).
  • the new Cas13 subtype protein and some known Cas13 proteins were subjected to multiple sequence alignment through MUSCLE software.
  • the alignment results were used to construct an evolutionary tree through FastTree software, and finally the evolutionary tree was presented through the ITOL website ( Figure 3).
  • Cas13i has the CRISPR conserved proteins Cas1 and Cas2, while the CRISPR Loci corresponding to other Cas13 subtypes lack these conserved proteins ( Figure 4).
  • DR direct repeat sequences
  • Table 1 for specific DR sequence information.
  • the secondary structure of the DR sequence of the new Cas13 subtype was predicted through the RNAfold website, showing that the predicted structure is also similar to the previous structures of Cas13a and Cas13b ( Figure 8).
  • the spacer corresponding to the new Cas13 subtype was predicted through CRISPRTarget, and some of the sequences were compared to the IMG/VR database, indicating that the new Cas13 subtype protein may help microorganisms defend against foreign mobile genetic factors.
  • the coding sequence of Cas13 was codon optimized (human) and synthesized.
  • the synthesized Cas13 effector protein sequence was inserted into the XmaI and NheI restriction enzyme sites of the pCAG-2A-eGFP vector to construct the pCAG-Cas13-2A-eGFP plasmid.
  • the protein sequences used are shown in Table 2.
  • the synthesized crRNA sequence (U6 promoter sequence + DR sequence + spacer sequence or U6 promoter sequence + spacer sequence + DR sequence) is inserted between the EcoRI and HindIII restriction enzyme sites of the pUC19-U6 vector to construct pUC19-U6 -5'-DR crRNA or pUC19-U6-3'-DR crRNA plasmid.
  • the corresponding crRNA targets mCherry mRNA, and the mCherry spacer sequence information is shown in Table 3.
  • the CAG promoter expresses eBFP protein and the EF1a promoter expresses mCherry protein to construct pCAG-eBFP-EF1a-mCherry plasmid ( Figure 11A).
  • HEK293T cells were cultured in DMEM (Gibco) medium containing 10% FBS (Gibco) and 1% Penicillin-Streptomycin (Gibco). Cells were digested with 0.25% Trypsin-EDTA (Gibco), transferred to a 24-well plate and cultured for 12 hours. When the cell density reached 90%, LIPOFECTAMINE 3000 Reagent (Invitrogen) was used for transfection.
  • Each well of the experimental group was transfected with 600ng pCAG-Cas13-2A-eGFP plasmid and 300ng pUC19-U6-5'-DR crRNA plasmid or pUC19-U6-3'-DR crRNA plasmid, and 10ng pCAG-eBFP-EF1a-mCherry plasmid.
  • the control group was only transfected with 600ng pCAG-Cas13-2A-eGFP plasmid and 10ng pCAG-eBFP-EF1a-mCherry plasmid. After 48 hours, the cells were digested with 0.25% Trypsin-EDTA (Gibco), and the obtained suspension cells were subjected to fluorescence-activated cell sorting (FACS) (Fig. 11A).
  • mCherry expression was analyzed by FACS 2 days after transfection. Compare 5'-DR crRNA and 3'-DR Regarding the mCherry expression corresponding to crRNA, it was found that the knockdown efficiency of 3'-DR crRNA corresponding to Cas13g1, Cas13h1, Cas13i1, Cas13j1, and Cas13k1 was higher than that of 5'-DR crRNA. It is proved that Cas13g-k corresponds to the crRNA direction of 3'-DR ( Figure 12). The specific editing result information is shown in Table 4. Figure 12 Each group had 3 biological replicates, and statistical significance was evaluated using a two-tailed t-test.
  • Example 4 Screening novel Cas13 proteins with high editing efficiency by targeting genes in vivo or in vitro
  • the coding sequence of Cas13 was codon optimized (human) and synthesized.
  • the synthesized Cas13 effector protein sequence was inserted into the XmaI and NheI restriction sites of the pCAG-2A-eGFP vector to construct the pCAG-Cas13-2A-eGFP plasmid.
  • the protein sequences used are shown in Table 2.
  • the synthesized crRNA sequence (U6 promoter sequence + spacer sequence + DR sequence) was inserted between the EcoRI and HindIII restriction sites of the pUC19-U6 vector to construct the pUC19-U6-3'-DR crRNA plasmid.
  • the spacer sequence information is shown in Table 3.
  • the CAG promoter expresses eBFP protein and the EF1a promoter expresses mCherry protein to construct pCAG-eBFP-EF1a-mCherry plasmid ( Figure 11A).
  • HEK293T cells were cultured in DMEM (Gibco) medium containing 10% FBS (Gibco) and 1% Penicillin-Streptomycin (Gibco). The cells were digested with 0.25% Trypsin-EDTA (Gibco), transferred to a 24-well plate and cultured for 12 hours. When the cell density reached 90%, LIPOFECTAMINE 3000 Reagent (Invitrogen) was used for transfection. Each well of the experimental group was transfected with 600ng pCAG-Cas13-2A-eGFP plasmid, 300ng pUC19-U6-3'-DR crRNA plasmid, and 10ng pCAG-eBFP-EF1a-mCherry plasmid.
  • the control group was only transfected with 600ng pCAG-Cas13-2A-eGFP plasmid and 10ng pCAG-eBFP-EF1a-mCherry plasmid.
  • 600ng pCAG-Cas13-2A-eGFP plasmid, 300ng non-targeting (NT) U6-crRNA plasmid and 10ng pCAG-eBFP-EF1a-mCherry plasmid were co-transfected into HEK293T cells as a non-targeting pair Photo group.
  • the cells were digested with 0.25% Trypsin-EDTA (Gibco), and the obtained suspension cells were subjected to fluorescence-activated cell sorting (FACS) (Fig. 11A).
  • mCherry expression was analyzed by flow cytometry 2 days after transfection.
  • the mean fluorescence intensity (MFI) of EGFP and mCherry double-positive cells in the experimental group was normalized to that of the control group.
  • Preliminary screening of Cas13 proteins with high editing efficiency (Figure 13A), among which the knockdown efficiency of RfxCas13d, Cas13X1, Cas13bt1-8, Cas13bt1-10, Cas13bt1-11, Cas13bt1-14, Cas13bt1-15, and Cas13g3 was higher than 50%. These proteins were used For the next experiment, the specific editing result information is shown in Table 5A.
  • HEK293T cells were cultured in DMEM (Gibco) medium containing 10% FBS (Gibco) and 1% Penicillin-Streptomycin (Gibco). Cells were digested with 0.25% Trypsin-EDTA (Gibco), transferred to a 24-well plate and cultured for 12 hours. When the cell density reached 90%, LIPOFECTAMINE 3000 Reagent (Invitrogen) was used for transfection. Each well of the experimental group was transfected with 600ng pCAG-Cas13-2A-eGFP plasmid and 300ng pUC19-U6-3'-DR crRNA plasmid.
  • the control group was only transfected with 600ng pCAG-Cas13-2A-eGFP plasmid ( Figure 11B).
  • the non-target control group was transfected with 600ng pCAG-Cas13-2A-eGFP plasmid and 300ng non-target U6-crRNA plasmid.
  • RNA quantitative analysis RNA quantitative analysis. RT-qPCR results were analyzed using the 2 - ⁇ CT method. The difference in the average CT values of the target gene and the internal reference gene GAPDH in three biological replicates was used to calculate the relative expression of the target gene, and the relative expression of the control group was used for analysis. standardization.
  • the coding sequence of the Cas13d homolog was codon optimized (human) and synthesized.
  • the synthesized Cas13 effector protein sequence was inserted into the XmaI and NheI restriction sites of the pCAG-2A-eGFP vector to construct the pCAG-Cas13d-2A-eGFP plasmid.
  • the protein sequences used are shown in Table 2.
  • the synthesized crRNA sequence (U6 promoter sequence + DR sequence + spacer sequence) was inserted between the EcoRI and HindIII restriction sites of the pUC19-U6 vector to construct the pUC19-U6-5'-DR crRNA plasmid.
  • the spacer sequence information is shown in Table 3.
  • HEK293T cells were cultured in DMEM (Gibco) medium containing 10% FBS (Gibco) and 1% Penicillin-Streptomycin (Gibco). Digest the cells with 0.25% Trypsin-EDTA (Gibco), transfer them to a 24-well plate and culture them for 12 hours. When the cell density reaches 90%, use LIPOFECTAMINE 3000 Reagent (Invitrogen) for transfection. Each well of the experimental group was transfected with 600ng pCAG-Cas13d-2A-eGFP plasmid and 300ng pUC19-U6-5'-DR crRNA plasmid. The control group was only transfected with 600ng pCAG-Cas13d-2A-eGFP plasmid ( Figure 11B).
  • RNA quantitative analysis RNA quantitative analysis. RT-qPCR results were analyzed using the 2 - ⁇ CT method. The difference in average CT values of the target gene and the internal reference gene GAPDH in three biological replicates was used to calculate the relative expression of the target gene, and the relative expression of the control group was calculated. Standardize the amount.
  • HEK293T cells were cultured in DMEM (Gibco) medium containing 10% FBS (Gibco) and 1% Penicillin-Streptomycin (Gibco). Digest the cells with 0.25% Trypsin-EDTA (Gibco), transfer them to a 24-well plate and culture them for 12 hours. When the cell density reaches 90%, use LIPOFECTAMINE 3000 Reagent (Invitrogen) for transfection. Each well of the experimental group was transfected with 600ng pCAG-Cas13-2A-eGFP plasmid and 300ng pUC19-U6-3'-DR crRNA plasmid, with crRNA targeting ANXA4 mRNA. The control group was only transfected with 600ng pCAG-Cas13-2A-eGFP plasmid.
  • HEK293T cells were cultured in DMEM (Gibco) medium containing 10% FBS (Gibco) and 1% Penicillin-Streptomycin (Gibco). Digest the cells with 0.25% Trypsin-EDTA (Gibco), transfer them to a 24-well plate and culture them for 12 hours. When the cell density reaches 90%, use LIPOFECTAMINE 3000 Reagent (Invitrogen) for transfection.
  • Each well of the cell viability experimental group was transfected with 600ng pCAG-Cas13-2A-eGFP plasmid and 300ng pUC19-U6-3'-DR crRNA plasmid, with crRNA targeting ANXA4 mRNA.
  • the control group was not transfected with any plasmid.
  • Calcein-AM/PI double strain kit (Solarbio) was used to measure fluorescent cell viability of HEK293T 2 days after transfection. Total cell quantification was performed at 490nm excitation and 515nm emission, and dead cell quantification was performed at 535nm excitation and 617nm emission. Measurements were made using flow cytometry.
  • Each well of the trans-cleavage experimental group was transfected with 600ng pCAG-Cas13-2A-eGFP plasmid, 10ng pCAG-eBFP-EF1a-mCherry plasmid and 300ng pUC19-U6-3'-DR crRNA plasmid, in which crRNA targets mCherry mRNA.
  • the trans-cleavage control group was transfected with non-targeting U6-crRNA plasmid.
  • the cis-cleaving ability of Cas13 protein was calculated by measuring the fluorescence intensity of mCherry, and the trans-cleaving ability was calculated by measuring the fluorescence intensity of EGFP.
  • HEK293T cells were cultured in DMEM (Gibco) medium containing 10% FBS (Gibco) and 1% Penicillin-Streptomycin (Gibco). Digest the cells with 0.25% Trypsin-EDTA (Gibco), transfer them to a 24-well plate and culture them for 12 hours. When the cell density reaches 90%, use LIPOFECTAMINE 3000 Reagent (Invitrogen) for transfection.
  • U6-crRNA plasmids targeting mCherry mRNA with spacer lengths ranging from 5 to 50nt.
  • spacer length is between 15-30nt
  • a crRNA is designed every 1 nt; when the spacer length is 5-15nt and 30-50nt, a crRNA is designed every 5nt.
  • 600ng pCAG-Cas13-2A-eGFP plasmid, 10ng pCAG-eBFP-EF1a-mCherry plasmid and 300ng pUC19-U6-3'-DR crRNA plasmid were co-transfected into HEK293T cells as the experimental group.
  • 600ng pCAG-Cas13-2A-eGFP plasmid, 300ng non-targeting U6-crRNA plasmid and 10ng pCAG-eBFP-EF1a-mCherry plasmid were co-transfected into HEK293T cells as a control group.
  • the MFI of EGFP and mCherry double-positive cells in the experimental group was standardized with the negative control.
  • the normalized results can reflect the impact of different spacer lengths on RNA interference.
  • the results showed that when using a spacer with a length of 27nt, Cas13g3 had the highest average knockout efficiency at the two target sites (Figure 19). See Table 10 for specific editing result information.
  • targets of 16 PFS combinations were cloned upstream of the mCherry gene of the EF-1 ⁇ -mCherry plasmid.
  • the MFI of EGFP and mCherry double-positive cells in the experimental group was standardized with the MFI of the negative control.
  • the experimental group was transfected with 600ng pCAG-Cas13-2A-eGFP plasmid, 10ng pCAG-eBFP-EF1a-mCherry plasmid and 300ng pUC19-U6-3'-DR.
  • crRNA plasmid HEK293T cells the negative control is HEK293T cells transfected with 600ng pCAG-Cas13-2A-eGFP plasmid, 300ng non-targeting U6-crRNA plasmid and 10ng pCAG-eBFP-EF1a-mCherry plasmid.
  • the results showed that Cas13g3 protein demonstrated stable and efficient interference effects in these 16 PFS sequences ( Figure 20).
  • the specific editing result information is shown in Table 11 (the spacer sequence is: SEQ ID NO.81:GCCGGCACTGATGAGGGCTGCCTAATT).
  • the optimal Cas13g3 editor composition is a crRNA plasmid with a spacer length of 27nt and a Cas13g3 protein plasmid fused with the NLS sequence.
  • HEK293T cells were cultured in DMEM (Gibco) medium containing 10% FBS (Gibco) and 1% Penicillin-Streptomycin (Gibco). Digest the cells with 0.25% Trypsin-EDTA (Gibco), transfer them to a 24-well plate and culture them for 12 hours. When the cell density reaches 90%, use LIPOFECTAMINE 3000 Reagent (Invitrogen) for transfection. Each well of the experimental group was transfected with 600ng pCAG-Cas13-2A-eGFP plasmid and 300ng pUC19-U6-3'-DR crRNA plasmid. The non-target control group was transfected with 600ng pCAG-Cas13-2A-eGFP plasmid and 300ng non-target U6-crRNA plasmid.
  • RNA quantitative analysis RNA quantitative analysis. RT-qPCR results were analyzed using the 2- ⁇ CT method. The difference in the average CT values of the target gene and the internal reference gene GAPDH in three biological replicates was used to calculate the relative expression of the target gene, and the relative expression of the control group was used for analysis. standardization.
  • Cas13X1, RfxCas13d and Cas13g3 can exhibit powerful RNA interference activity on EZH2, NF2, HRAS, NRAS and PPARG genes, and their average knockdown efficiencies are respectively 52.84%, 54.58% and 60.37% (Figure 22).
  • the specific editing results are shown in Table 13. Paired-t-test statistical analysis of the interference results showed that Cas13g3 has efficient RNA knockdown activity, which is comparable to Cas13X1 and RfxCas13d.

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Abstract

涉及经分离的新型CRISPR/Cas13蛋白，基于它的基因编辑系统以及使用它们进行RNA水平基因编辑的方法。提供了非天然存在或经工程化改造的RNA靶向系统，这些系统含有一个靶向RNA的新型Cas13效应蛋白，以及至少一种向导分子。

Description

经分离的Cas13蛋白、基于它的基因编辑系统及其用途 技术领域
本申请为生物技术领域，具体涉及经分离的Cas13蛋白，基于它的基因编辑系统以及使用它们进行RNA水平基因编辑的方法。
背景技术
基因编辑(gene editing)技术使得DNA序列定位点进行改造成为可能，比如第一代基因编辑工具锌指核酸酶(zinc finger nucleases,ZFNs)，第二代基因编辑工具类似转录激活的小型核酸酶(transcription activator-like effector nucleases,TALENs)，第三代基因编辑工具中的II型及V型的成簇的规律间隔的短回文重复序列(CRISPR,Clustered Regularly Interspaced Short Palindromic Repeat)/Cas(CRISPR-associated protein)都可以用于靶向改造基因组，但这些基因编辑系统只能靶向基因组及外来的DNA，而不能对体内RNA进行靶向编辑。
VI型的CRISPR/Cas13系统，是来自古生菌和细菌的天然免疫系统。其不同于以往基因编辑工具，利用核酸碱基互补配对原理进行目标RNA序列的识别，引导Cas效应蛋白进行靶向切割，适用性强、设计简单、效率高。
其中，VI-D型CRISPR/Cas13基因编辑系统是现下应用较为广泛的VI型CRISPR/Cas系统。这一系统在原核生物中可通过识别并切割靶向的多核苷酸上双侧的原间隔序列侧翼位点(Protospacer flanking site,PFS)序列，从而对单链RNA进行靶向编辑，而在真核生物中，VI-D型CRISPR/Cas系统对RNA的靶向编辑作用无PFS序列。同时相比于其他VI型系统，VI-D型系统具有更小的蛋白体积，在基于腺相关病毒(Adeno-associated virus,AAV)的基因治疗具有更好的前景。在庞大以及各色各样的宏基因组中，隐藏了尚未培养甚至尚未发现的微生物，可能存在大量的未被发现的VI型CRISPR/Cas13系统，在这些微生物中寻找和克隆获得更具优良特性的Cas蛋白是人们所期望的。
发明内容
本申请的目的就是在宏基因组中寻找新型Cas13基因编辑系统并开发其用途。具体地提供了：
1.经分离的Cas13核酸酶蛋白，所述Cas13蛋白的氨基酸序列包括：
a)如SEQ ID NO.1～28中任一项所示的氨基酸序列；或，
b)与SEQ ID NO.1～28中的任一项具有至少80％序列同一性、且具有RNA切割活性的氨基酸序列。
2.如项2所述的Cas13核酸酶蛋白，其中所述Cas13蛋白是Cas13bt1、Cas13bt2、Cas13g、Cas13h、Cas13i、Cas13j或Cas13k蛋白。
3.一种工程化的Cas13核酸酶效应蛋白，包含:
a)如SEQ ID NO.1～28中任一项所示的氨基酸序列；或，
b)与SEQ ID NO.1～28中的任一项具有80％以上序列同一性、且具有RNA切割活性的氨基酸序列。
4.项3的效应蛋白，其中所述Cas13核酸酶效应蛋白通过氨基酸突变失去催化活性，例如所述Cas13核酸酶蛋白在其C端、N端的HEPN结构域(RxxxxH基序)进行突变形成dCas13蛋白。
5.项3或4所述的效应蛋白，其还包含与所述Cas13核酸酶蛋白融合的功能结构域。
6.项5所述的效应蛋白，所述功能结构域选自如下的一项或多项：翻译起始结构域、翻译阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域、逆转录酶结构域、报告分子结构域和核酸酶结构域。
7.项6所述的效应蛋白，其中所述核碱基编辑结构域为腺苷脱氨酶、胞苷脱氨酶或所述它们的催化结构域融合。
8.一种多核苷酸，其编码项1或2的Cas13核酸酶蛋白或项3-7任一项的效应蛋白。
9.包含如项8所述的多核苷酸的载体，优选所述载体为质粒或慢病毒。
10.一种工程化的CRISPR-Cas13基因编辑系统，包含：
(a)项3-7中任一项所述的工程化Cas13核酸酶效应蛋白或编码所述效应蛋白的核酸；以及
(b)crRNA，其包含与靶序列互补的间隔区序列，当分别使用如SEQ ID NO.1～28所示的工程化Cas13核酸酶效应蛋白时所述crRNA还分别包含与 SEQ ID NO.29～56所示的序列具有至少80％同一性的直接重复(direct repeat,DR)序列；
其中所述工程化的Cas13核酸酶效应蛋白和所述crRNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合包含所述靶序列的靶核酸并诱导所述靶核酸的修饰。
11.包含如项10所述的工程化的CRISPR-Cas13基因编辑系统的试剂盒。
12.如项3-7任一项所述的工程化的CRISPR-Cas13核酸酶效应蛋白、项10的工程化的CRISPR-Cas13基因编辑系统在制备治疗与个体的细胞中与核酸突变相关的疾病或病症的药物中的用途。
13.一种修饰细胞中包含的靶核酸的方法，包括使所述细胞与项3-7任一项所述的工程化的CRISPR-Cas13核酸酶蛋白效应蛋白、项10的工程化的CRISPR-Cas13基因编辑系统接触从而实现对细胞中所述靶核酸的修饰。
14.如项3-7任一项所述的工程化的CRISPR-Cas13核酸酶效应蛋白或项10的工程化的CRISPR-Cas13基因编辑系统的用于制备用来治疗个体中与核酸突变相关的疾病或病症的药物的用途。
本申请还提供了：
1.经分离的Cas13核酸酶蛋白，所述Cas13核酸酶蛋白的氨基酸序列为：
a)如SEQ ID NO.1～28中任一序列所示的氨基酸序列；或，
b)与SEQ ID NO.1～28中的任一序列具有至少80％的序列同一性、且具有RNA切割活性的氨基酸序列。
2.如项1所述的Cas13核酸酶蛋白，其中所述Cas13核酸酶蛋白是Cas13bt1、Cas13bt2、Cas13g、Cas13h、Cas13i、Cas13j或Cas13k蛋白，优选为Cas13g3。
3.一种工程化的Cas13核酸酶效应蛋白，包含Cas13核酸酶蛋白，该Cas13核酸酶蛋白包含:
a)如SEQ ID NO.1～28中任一序列所示的氨基酸序列；或，
b)与SEQ ID NO.1～28中的任一序列具有80％以上序列同一性、且具有RNA切割活性的氨基酸序列。
4.如项3的所述效应蛋白，其中所述Cas13核酸酶蛋白通过氨基酸突变失去催化活性，例如所述Cas13核酸酶蛋白在其C端和/或N端的HEPN结构域(RxxxxH基序)通过氨基酸突变形成dCas13蛋白。
5.项3或4所述的效应蛋白，其还包含与所述Cas13核酸酶蛋白融合的功能结构域。
6.如项5所述的效应蛋白，所述功能结构域选自如下的一项或多项：翻译起始结构域、翻译阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域、逆转录酶结构域、报告分子结构域和核酸酶结构域。
7.如项6所述的效应蛋白，其中所述核碱基编辑结构域为腺苷脱氨酶、胞苷脱氨酶或它们的催化结构域。
8.一种多核苷酸，其编码项1或2的Cas13核酸酶蛋白或项3-7任一项的效应蛋白。
9.包含如项8所述的多核苷酸的载体，所述载体为质粒或病毒，所述病毒优选为慢病毒。
10.一种工程化的CRISPR-Cas13基因编辑系统，包含：
(a)如项1或2所述的核酸酶、如项3-7中任一项所述的工程化Cas13核酸酶效应蛋白或编码所述效应蛋白的核酸；以及
(b)crRNA，其包含与靶核酸中的靶序列互补的间隔区序列；
其中所述工程化的Cas13核酸酶效应蛋白和所述crRNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合包含所述靶序列的靶核酸并诱导所述靶核酸的修饰。
11.如项10所述的CRISPR-Cas13基因编辑系统，其中所述crRNA还包含直接重复(DR)序列，优选所述直接重复(DR)序列包含SEQ ID NO.29～56中任一序列所示的序列，或包含与SEQ ID NO.29～56中任一序列所示的序列具有至少80％同一性的序列。
12.包含如项10或11所述的工程化的CRISPR-Cas13基因编辑系统的试剂盒。
13.如项1或2所述的Cas13核酸酶蛋白、如项3-7中任一项所述的工程化的CRISPR-Cas13核酸酶效应蛋白、如项10或11所述的工程化的CRISPR-Cas13基因编辑系统在制备治疗与个体的细胞中与核酸突变相关的疾病或病症的药物中的用途。
14.一种修饰细胞中包含靶核酸的方法，包括使所述细胞与如项1或2所述的Cas13核酸酶蛋白、如项3-7任一项所述的工程化的Cas13核酸酶效 应蛋白、如项10或11所述的工程化的CRISPR-Cas13基因编辑系统接触，从而实现对所述细胞中所述靶核酸的修饰。
15.包含如项1或2所述的Cas13核酸酶蛋白、如项3-7任一项所述的工程化的Cas13核酸酶效应蛋白或如项10或11所述的工程化的CRISPR-Cas13基因编辑系统的组合物，其用于核酸的修饰。
本申请的技术方案实现的有益技术效果
本申请通过对宏基因组数据进行分析，最后鉴定出5个全新亚型共75个Cas13蛋白。全新Cas13亚型命名为Cas13g-k；其中Cas13g蛋白质大小约为800个氨基酸，Cas13h-k蛋白大小约为1100个氨基酸。这些新型蛋白都体现了良好的RNA水平的靶向切割活性。我们报道新发现的Cas13g3蛋白可以设计成可编程的RNA编辑。Cas13g3体积为767aa，被认为是目前效率最高的小Cas13蛋白，可被用作有前途的哺乳动物RNA编辑工具。
附图说明
图1示出了通过对宏基因组数据进行分析，最后鉴定出5个全新亚型共75个Cas13蛋白的流程图。
图2示出了7个亚型与4个旧亚型蛋白同源性以及每种亚型蛋白平均大小的示意图。
图3示出了7个亚型与4个旧亚型蛋白同源性的另一示意图。
图4示出了Cas13bt1-Cas13k的Loci位点的间隔-定向重复序列的示意图。
图5示出了Cas13bt1-Cas13k的在C端、N端的HEPN结构域的示意图。
图6示出了Cas13bt1-Cas13k的RxxxxH基序的比例。
图7为Cas13bt1-Cas13k的在C端、N端的HEPN结构域的RxxxxH基序序列保守性示意图。
图8示出了Cas13bt1-Cas13k的DR序列预测结构的示意图。
图9示出了Cas13a-Cas13k的在各种环境的微生物中的分布比例。
图10示出了Cas13a-Cas13k的在自然界的存在比例。
图11A和11B为分析HEK293T细胞中Cas13蛋白RNA切割活性的实验过程的示意图。
图12为使用3'-DR或5'-DR-crRNA敲低mCherry mRNA的效率比较图。
图13A和13B为敲低mCherry mRNA筛选编辑效率高的Cas13蛋白的 结果图。
图14A和14B为新型Cas13介导的ANXA4 mRNA敲除的实时定量逆转录PCR(real time quantitative reverse transcription PCR,RT-qPCR)结果图。
图15示出了RfxCas13d、Cas13X1、Cas13bt1-11和Cas13g3介导的内源mRNA敲低的定量RT-qPCR结果图。
图16为新型Cas13d介导的ANXA4 mRNA敲低的RT-qPCR结果图。
图17示出了RfxCas13d、Cas13X1、Cas13bt1-11和Cas13g3介导的ANXA4 mRNA敲低的RNA-seq分析结果图。
图18A和18B示出了RfxCas13d、Cas13X1、Cas13bt1-11和Cas13g3的反式活性评估。
图19A和19B示出了Cas13g3系统最优spacer长度分析。
图20示出了Cas13g3系统PFS偏好性分析。
图21示出了NLS对Cas13g3系统干扰活性的影响。
图22示出了Cas13g3系统与其他Cas13系统RNA干扰能力的比较。
具体实施方式
下面将参照附图更详细地描述本发明的具体实施例。虽然附图中显示了本发明的具体实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
需要说明的是，在说明书及权利要求当中使用了某些词汇来指称特定组件。本领域技术人员应可以理解，技术人员可能会用不同名词来称呼同一个组件。本说明书及权利要求并不以名词的差异来作为区分组件的方式，而是以组件在功能上的差异来作为区分的准则。如在通篇说明书及权利要求当中所提及的“包含”或“包括”为一开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本发明的较佳实施方式，然所述描述乃以说明书的一般原则为目的，并非用以限定本发明的范围。本发明的保护范围当视所附权利要求所界定者为准。
如本文所用，就特定组分而言“基本上不含”在本文中用于表示特定组分未被有目的地配制到组合物中和/或仅作为污染物或以痕量存在。因此，由组合物的任何意外污染导致的特定组分的总量低于0.05％，优选低于0.01％。 最优选的是其中特定组分的量用标准分析方法检测不到的组合物。
如在本说明书中所使用的，“一”或“一个”可以表示一个或多个。如权利要求中所使用的，当与单词“包含”一起使用时，单词“一”或“一个”可以表示一个或多于一个。
在权利要求中使用术语“或”用于表示“和/或”，除非明确指出仅指代替代方案或者替代方案是相互排斥的，尽管本公开内容支持仅指代替代方案和“和/或”的定义。如本文所用，“另一个”可以表示至少第二个或更多个。
贯穿本申请，术语“约”用于指示值包括装置的误差的固有变化，该方法用于测定该值或存在于研究对象之间的变化。
本申请在第一方面提供了一种经分离的Cas13核酸酶蛋白。
在一个具体实施方式中，提供了一种经分离的Cas13核酸酶蛋白，所述Cas13蛋白的氨基酸序列包括：a)如SEQ ID NO.1～28中任一项所示的氨基酸序列；或，b)与SEQ ID NO.1～28中的任一项具有80％以上序列同一性、且具有RNA切割活性的氨基酸序列。在又一具体实施方式中，提供了一种Cas13核酸酶蛋白，其中所述Cas13蛋白是Cas13bt1、Cas13bt2、Cas13g、Cas13h、Cas13i、Cas13j或Cas13k蛋白。
在本说明书的上下文中，术语Cas13a和C2c2可以互换使用。它最初是由Broad研究所的研究人员发现的。2015年，Shmakov及其同事利用Cas1作为“诱饵”，来鉴定细菌基因组中与CRISPR相关联的蛋白。通过此次分析，他们鉴定出53个候选基因，总共分成三大类：C2c1、C2c2和C2c3。C2c1和C2c3与Cpf1有些类似，不过它们需要tracrRNA和crRNA才能切割DNA目标，而Cpf1仅需要crRNA。而C2c2(也就是Cas13a)与Cas9的区别在于，Cas13a结合并切割RNA，而Cas9切割DNA。Cas9利用tracrRNA和crRNA来实现与DNA目标的结合和切割。而Cas13a只需要24个碱基的crRNA，它通过富含尿嘧啶的茎环结构与Cas13a分子相互作用，并通过Cas13a的一系列构象变化来促进目标的切割。与Cas9一样，Cas13a也能容忍crRNA与目标序列之间的单个错配，不过若存在2个错配，那切割效率就大大降低。它的PFS序列(相当于PAM序列)位于间隔区的3'端，由A、U或C碱基组成。Cas13a还有一个特别之处，那就是Cas13a一旦识别并切割由crRNA序列指定的RNA目标，它就转入了一种酶促“激活”状态，这时 它将结合并切割其他的RNA，而不管它们是否与crRNA同源，或是否存在PFS；这一点与Cas9截然不同。在Cas13系统中不需要tracrRNA，只需要crRNA；而crRNA由DR序列(即直接重复序列)和spacer序列(间隔序列，即与靶序列互补的核苷酸序列)构成。
本申请在第二方面提供了一种工程化的Cas13核酸酶效应蛋白
在一个具体实施方式中，提供了一种工程化的Cas13核酸酶效应蛋白，包含:a)如SEQ ID NO.1～28中任一项所示的氨基酸序列；或，b)与SEQ ID NO.1～28中的任一项具有80％以上序列同一性、且具有RNA切割活性的氨基酸序列。优选所述核酸酶效应蛋白与与SEQ ID NO.1～28中的任一项具有80％以上、81％以上、82％以上、83％以上、84％以上、85％以上、86％以上、87％以上、88％以上、89％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上、或100％的序列同一性。
在一个具体实施方式中，提供了一种工程化的Cas13核酸酶效应蛋白，所述Cas13核酸酶效应蛋白通过氨基酸突变失去催化活性，例如所述Cas13核酸酶蛋白在其C端、N端的HEPN结构域(RxxxxH基序)进行突变形成dCas13蛋白。
在本说明书的上下文中，术语HEPN是指Cas13a含有的两个结构域，它对于Cas13的作用相当于HNH和RuvC结构域对于Cas9的作用，是来切割目标核酸。HEPN结构域对Cas13a切割RNA目标是必需的。HEPN核糖核酸酶结构域的RxxxxH基序分别位于Cas13蛋白质的N端和C端。另外，与Cas9类似，Cas13a分子中关键残基的突变可以形成核酸酶活性缺失的Cas13a(dCas13a)，它能够结合RNA目标但无法切割。RxxxxH基序为6个连续氨基酸，第一个氨基酸为精氨酸R，最后一个为组氨酸H，中间可以是任意氨基酸。RxxxxH基序即HEPN基序，具有核糖核酸酶功能。
在一个具体实施方式中，提供了一种工程化的Cas13核酸酶效应蛋白，其还包含与所述Cas13核酸酶蛋白融合的功能结构域。其中，所述功能结构域选自如下的一项或多项：翻译起始结构域、翻译阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域、逆转录酶结构域、报告分子结构域和核酸酶结构域；其中所述核碱基编辑结构域为腺苷脱氨酶、胞苷 脱氨酶或所述它们的催化结构域融合。
本申请在第三方面涉及一种多核苷酸。
在一个具体实施方式中，提供了一种多核苷酸，其编码前述Cas13核酸酶蛋白或前述工程化效应蛋白。
本申请在第四方面涉及一种多核苷酸的载体。
在一个具体实施方式中，提供了一种多核苷酸的载体，优选所述载体为质粒或慢病毒。
本申请在第五方面涉及一种基因编辑系统。
在一个具体实施方式中，提供了一种工程化的CRISPR-Cas13基因编辑系统，包含：(a)前述的工程化Cas13核酸酶效应蛋白或编码所述效应蛋白的核酸；以及(b)crRNA，其包含与靶序列互补的间隔区序列，当分别使用如SEQ ID NO.1～28所示的工程化Cas13核酸酶效应蛋白时所述crRNA还分别包含与SEQ ID NO.29～56所示的序列具有至少80％同一性的直接重复(DR)序列；其中所述工程化的Cas13核酸酶效应蛋白和所述crRNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合包含所述靶序列的靶核酸并诱导所述靶核酸的修饰。优选所述直接重复(DR)序列与SEQ ID NO.29～56所示的序列具有至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
本申请在第六方面涉及一种试剂盒。
在一个具体实施方式中，提供了包含前述工程化的CRISPR-Cas13基因编辑系统的试剂盒。
本申请在第七方面涉及工程化CRISPR-Cas13核酸酶效应蛋白的用途。
在一个具体实施方式中，提供了前述的工程化的CRISPR-Cas13核酸酶效应蛋白、前述工程化的CRISPR-Cas13基因编辑系统在制备治疗与个体的细胞中与核酸突变相关的疾病或病症的药物中的用途。
本申请在第八方面涉及一种修饰细胞中包含的靶核酸的方法。
在一个具体实施方式中，提供了一种修饰细胞中包含靶核酸的方法，包括使所述细胞与前述的工程化的CRISPR-Cas13核酸酶蛋白效应蛋白、前述工程化的CRISPR-Cas13基因编辑系统接触，从而实现对所述细胞中所述靶核酸的修饰。
本申请在第九方面涉及一种治疗个体中与核酸突变相关的疾病或病症用途。
在一个具体实施方式中，提供了前述的工程化的CRISPR-Cas13核酸酶效应蛋白或前述工程化的CRISPR-Cas13基因编辑系统的用于制备用来治疗个体中与核酸突变相关的疾病或病症的药物的用途。
实施例部分
实施例1：鉴定Cas13新亚型
寻找新的Cas13
从JGI(Joint Genome Institute)数据库下载主要来自人类和动物宿主、水生、自然和农业土壤等近10Tb(Terabyte)组装的宏基因组数据。使用MinCED软件预测大约500251个CRISPR array，提取CRISPR array区域上下游20kb(kilobase)序列作为候选序列，对候选序列使用Meta-GeneMark 1进行蛋白质开放性阅读框(open reading frame,ORF)，得到2596558候选蛋白。鉴于已知Cas蛋白体积，我们将蛋白体积大于400个氨基酸的蛋白作为候选蛋白，获得395847个蛋白质。从NCBI(National Center for Biotechnology Information)中下载已知Cas13a、Cas13b、Cas13c和Cas13d序列，使用HH-suite3软件构建已知Cas13蛋白质数据库。将395847个候选蛋白与已知Cas13蛋白质数据库通过HH-suite3软件的hhsearch进行比对，获得具有比对结果的6400个蛋白。去除候选蛋白中不完整以及含有少于2个高等真核生物和原核生物的核苷酸结合(Higher eukaryotes and prokaryotes nuceotide-binding,HEPN)结 构域蛋白，得到2253个候选物。除去已知Cas13蛋白序列，得到1139个新Cas13(图1)。
系统发育分析鉴定全新Cas13亚型
对候选蛋白进行系统发育分析以及分类，2253个候选蛋白首先通过MMseqs软件的cluster--min-seq-id 0.5-c 0.7参数进行初步聚类得到聚类，之后将每个聚类通过mmseqs软件的cluster--min-seq-id 0.9-c 0.8参数去除冗余蛋白。之后将每个聚类通过MAFFT软件进行多序列比对，比对结果通过HH-suite3软件的hhmake构建隐马尔可夫模型，之后将隐马尔可夫模型文件通过HH-suite3软件的hhalign进行相互比对。蛋白质之间比对分数用来计算进化距离。其中sij表示i蛋白与j蛋白比对分数；sji表示j蛋白与i蛋白比对分数；Sij表示i蛋白与j蛋白平均比对分数；dij代表i蛋白与j蛋白的进化距离。进化距离计算方式为Sij＝(sij+sji)/2，dij＝(log(min(Sii,Sjj))-log(Sij))/2。进化距离结果通过非加权组平均法(UPGMA)进行聚类分析。最后鉴定出很多已知Cas13a-d同源物和7个不同分支。其中两个分支与之前鉴定的Cas13X、Cas13Y和Cas13bt同源，我们将它们命名为Cas13bt1和Cas13bt2。剩余5个分支为全新亚型，我们命名为Cas13g-k。其中Cas13g蛋白质大小约为800个氨基酸，Cas13h-k蛋白大小约为1100个氨基酸(图1、2)。
Cas13亚型进化树分析
全新Cas13亚型蛋白与部分已知Cas13蛋白通过MUSCLE软件进行多序列比对，比对结果通过FastTree软件构建进化树，最后通过ITOL网站呈现进化树(图3)。
实施例2：鉴定Cas13新亚型的特征
新Cas13亚型CRISPR loci分析
提取CRISPR array区域上下游20kb序列进行ORF预测，获得的蛋白序列使用blast软件进行比对。Cas13i具有CRISPR保守蛋白Cas1和Cas2，而其他Cas13亚型对应的CRISPR Loci缺乏这些保守蛋白(图4)。
Cas13亚型HEPN结构域分析
对每种亚型蛋白质序列通过MUSCLE软件进行多序列比对，比对结果提取出各个蛋白的HEPN结构域信息。绘制对应HEPN结构域在每个Cas13亚型的代表蛋白分布图(图5)。对每个Cas13亚型蛋白对应的RXXXXH基序进行 排序，展现前两种基序所占比例(图6)。最后对全新Cas13亚型蛋白的HEPN结构域保守性进行分析(图7)。
全新Cas13亚型CRISPR阵列分析
通过MinCED软件寻找直接重复序列(DR)，具体DR序列的信息见表1。通过RNAfold网站对全新Cas13亚型的DR序列的二级结构进行预测，显示其预测的结构也与之前Cas13a和Cas13b结构类似(图8)。全新Cas13亚型对应的spacer通过CRISPRTarget进行预测，其中部分序列比对到IMG/VR数据库，说明全新Cas13亚型蛋白可能帮助微生物防御外来可动遗传因子。
表1
新型Cas13分布
将所有新型Cas13(1139个)分类情况以及宏基因组来源进行统计发现，80％Cas13d来源于人体微生物，Cas13c都来自于水源，超过30％Cas13a来自人体微生物或水源，Cas13b分布较分散。其中Cas13d主要分布在动物微生物 中，与之前发现其主要分布在革兰氏阳性菌中的瘤胃球菌属一致。对应新型Cas亚型，Cas13i分布于人体或动物体内，Cas13g主要分布于富含甲烷环境中(图9)。同时所有新型Cas13(1139个)中Cas13a、Cas13b、Cas13d分别占总量的23.7％、46.2％、9.5％(图10)。
实施例3：鉴定新型Cas13亚型crRNA方向
质粒构建
Cas13的编码序列通过密码子优化(人类)并合成。合成的Cas13效应蛋白序列插入到pCAG-2A-eGFP载体的XmaI和NheI限制性酶切位点中，构建pCAG-Cas13-2A-eGFP质粒，使用到的蛋白质序列见表2。合成的crRNA序列(U6启动子序列+DR序列+spacer序列或U6启动子序列+spacer序列+DR序列)插入到pUC19-U6载体的EcoRI和HindIII限制性酶切位点之间，构建pUC19-U6-5'-DR crRNA或pUC19-U6-3'-DR crRNA质粒。其中对应的crRNA靶向mCherry mRNA，mCherry spacer序列信息见表3。CAG启动子表达eBFP蛋白和EF1a启动子表达mCherry蛋白构建pCAG-eBFP-EF1a-mCherry质粒(图11A)。
表2







表3：

细胞培养，转染
HEK293T细胞在含有10％FBS(Gibco)和1％Penicillin-Streptomycin(Gibco)的DMEM(Gibco)培养基中培养。使用0.25％Trypsin-EDTA(Gibco)消化细胞，转入24孔板中培养12小时，等细胞密度达到90％时，使用LIPOFECTAMINE 3000 Reagent(Invitrogen)进行转染。实验组每个孔转染600ng pCAG-Cas13-2A-eGFP质粒和300ng pUC19-U6-5'-DR crRNA质粒或pUC19-U6-3'-DR crRNA质粒，以及10ng pCAG-eBFP-EF1a-mCherry质粒。对照组只转染600ng pCAG-Cas13-2A-eGFP质粒和10ng pCAG-eBFP-EF1a-mCherry质粒。48小时之后，使用0.25％Trypsin-EDTA(Gibco)消化细胞，获得的悬浮细胞进行荧光激活细胞分选(FACS)(图11A)。
靶向mCherry mRNA鉴定新型Cas13亚型crRNA方向
转染2天后通过FACS分析mCherry表达情况。比较5'-DR crRNA和3'-DR  crRNA对应的mCherry表达情况，发现Cas13g1、Cas13h1、Cas13i1、Cas13j1、Cas13k1对应的3'-DR crRNA敲低效率高于5'-DR crRNA。证明Cas13g-k对应crRNA方向为3'-DR(图12)，具体编辑结果信息见表4。图12每组都有3个生物学复本，使用双尾t-检验评价统计学意义。
表4：
实施例4：通过靶向体内基因或体外基因筛选编辑效率高的新型Cas13蛋白
质粒构建
Cas13的编码序列通过密码子优化(人类)并合成。合成的Cas13效应蛋白序列插入到pCAG-2A-eGFP载体的XmaI和NheI酶切位点中，构建pCAG-Cas13-2A-eGFP质粒，使用到的蛋白质序列见表2。合成的crRNA序列(U6启动子序列+spacer序列+DR序列)插入到pUC19-U6载体的EcoRI和HindIII酶切位点之间，构建pUC19-U6-3'-DR crRNA质粒。其中spacer序列信息见表3。CAG启动子表达eBFP蛋白和EF1a启动子表达mCherry蛋白构建pCAG-eBFP-EF1a-mCherry质粒(图11A)。
细胞培养，转染
HEK293T细胞在含有10％FBS(Gibco)和1％Penicillin-Streptomycin(Gibco)的DMEM(Gibco)培养基中培养。使用0.25％Trypsin-EDTA(Gibco)消化细胞，转入24孔板中培养12小时，等细胞密度达到90％时，使用LIPOFECTAMINE 3000 Reagent(Invitrogen)进行转染。实验组每个孔转染600ng pCAG-Cas13-2A-eGFP质粒和300ng pUC19-U6-3'-DR crRNA质粒，以及10ng pCAG-eBFP-EF1a-mCherry质粒。对照组只转染600ng pCAG-Cas13-2A-eGFP质粒和10ng pCAG-eBFP-EF1a-mCherry质粒。600ngpCAG-Cas13-2A-eGFP质粒、300ng非靶向(non-targeting,NT)的U6-crRNA质粒和10ng pCAG-eBFP-EF1a-mCherry质粒三者共转到HEK293T细胞中作为非靶标对 照组。48小时之后，使用0.25％Trypsin-EDTA(Gibco)消化细胞，获得的悬浮细胞进行荧光激活细胞分选(FACS)(图11A)。
敲低mCherry mRNA筛选编辑效率高的Cas13蛋白
转染2天后通过流式分析mCherry表达情况。实验组EGFP和mCherry双阳性细胞的平均荧光强度(mean fluorescence intensity,MFI)与对照组进行了标准化处理。初步筛选编辑效率高的Cas13蛋白(图13A)，其中RfxCas13d、Cas13X1、Cas13bt1-8、Cas13bt1-10、Cas13bt1-11、Cas13bt1-14、Cas13bt1-15、Cas13g3敲低效率高于50％，这些蛋白用于接下来实验，具体编辑结果信息见表5A。
转染2天后通过流式分析mCherry表达情况。实验组EGFP和mCherry双阳性细胞的MFI与非靶标对照组进行了标准化处理。MFI结果(图13B)和具体编辑结果信息见表5B。
表5A：
表5B：

细胞培养，转染
HEK293T细胞在含有10％FBS(Gibco)和1％Penicillin-Streptomycin(Gibco)的DMEM(Gibco)培养基中培养。使用0.25％Trypsin-EDTA(Gibco)消化细胞，转入24孔板中培养12小时，等细胞密度达到90％时，使用LIPOFECTAMINE 3000 Reagent(Invitrogen)进行转染。实验组每个孔转染600ng pCAG-Cas13-2A-eGFP质粒和300ng pUC19-U6-3'-DR crRNA质粒。对照组只转染600ng pCAG-Cas13-2A-eGFP质粒(图11B)。非靶标对照组转染600ng pCAG-Cas13-2A-eGFP质粒和300ng非靶标的U6-crRNA质粒。
RNA提取，RNA定量分析
转染48小时后使用Reagent(Life Technologie)裂解细胞，之后使用PureLinkTMRNA Mini Kit(Thermofisher)提取RNA。最后通过II One Step qRT-PCR SYBR Green Kit(Vazyme)进行RNA定量分析。RT-qPCR结果采用2-ΔΔCT法进行分析，利用靶基因与内参基因GAPDH在3个生物学复本中的平均CT值的差异计算靶基因的相对表达量，并以对照组的相对表达量进行标准化。
敲低体内基因ANXA4 mRNA筛选编辑效率高的Cas13蛋白
通过靶向ANXA4 mRNA与对照组的标准化RT-qPCR结果，发现Cas13bt1-11，Cas13bt1-15和Cas13g3具有较高编辑效率(图14A)，具体编辑结果信息见表6A。实验组与非靶标对照组的标准化RT-qPCR结果也显示Cas13bt1-8、Cas13bt1-11、Cas13bt1-15和Cas13g3具有较高编辑(图14B)，具体编辑结果信息见表6B。值得注意的是，两次实验都显示Cas13g3具有最强的RNA干扰能力和超小的蛋白质分子量(767个氨基酸)，我们选择Cas13g3蛋白进行进一步的特征分析。
表6A：
表6B：
敲低体内基因筛选编辑效率高的Cas13蛋白
通过靶向4个体内基因，发现RfxCas13d在STAT3和KRAS位点编辑效率高于Cas13g3，而在EGFR和CXCR4的编辑效果更低，整体来看，Cas13g3的编辑效果略低于RfxCas13d，但超过了Cas13X1(图15)，编辑结果见表7。
表7：
实施例5：通过靶向体内基因验证Cas13d同源物编辑效率
质粒构建
Cas13d同源物的编码序列通过密码子优化(人类)并合成。合成的Cas13效应蛋白序列插入到pCAG-2A-eGFP载体的XmaI和NheI酶切位点中，构建pCAG-Cas13d-2A-eGFP质粒，使用到的蛋白质序列见表2。合成的crRNA序列(U6启动子序列+DR序列+spacer序列)插入到pUC19-U6载体的EcoRI和HindIII酶切位点之间，构建pUC19-U6-5'-DR crRNA质粒。其中spacer序列信息见表3。
细胞培养，转染
HEK293T细胞在含有10％FBS(Gibco)和1％Penicillin-Streptomycin(Gibco)的DMEM(Gibco)培养基中培养。使用0.25％Trypsin-EDTA(Gibco)消化细胞，转入24孔板中培养12小时，等细胞密度达到90％时，使用LIPOFECTAMINE 3000 Reagent(Invitrogen)进行转染。实验组每个孔转染600ng pCAG-Cas13d-2A-eGFP质粒和300ng pUC19-U6-5'-DR crRNA质粒。对照组只转染600ng pCAG-Cas13d-2A-eGFP质粒(图11B)。
RNA提取，RNA定量分析
转染48小时后使用Reagent(Life Technologie)裂解细胞，之后使用PureLinkTMRNA Mini Kit(Thermofisher)提取RNA。最后通过II One Step qRT-PCR SYBR Green Kit(Vazyme)进行RNA定量分析。RT-qPCR结果采用2-ΔΔCT法进行分析，利用靶基因与内参基因GAPDH在3个生物学复本中的平均CT值的差异计算靶基因的相对表达量，并以对照组的相对表 达量进行标准化。
敲低体内基因ANXA4 mRNA筛选编辑效率高的Cas13蛋白
通过靶向ANXA4 mRNA的RT-qPCR结果，发现Cas13d_968aa，Cas13d_982aa和Cas13d_999aa具有较高编辑效率(图16)，具体编辑结果信息见表8。
表8：
实施例6：使用RNA-seq探究Cas13的脱靶活性
细胞培养，转染
HEK293T细胞在含有10％FBS(Gibco)和1％Penicillin-Streptomycin(Gibco)的DMEM(Gibco)培养基中培养。使用0.25％Trypsin-EDTA(Gibco)消化细胞，转入24孔板中培养12小时，等细胞密度达到90％时，使用LIPOFECTAMINE 3000 Reagent(Invitrogen)进行转染。实验组每孔转染600ng pCAG-Cas13-2A-eGFP质粒和300ng pUC19-U6-3'-DR crRNA质粒，crRNA靶向ANXA4 mRNA。对照组只转染600ng pCAG-Cas13-2A-eGFP质粒。
RNA-seq
转染2天后使用Reagent(Life Technologie)裂解细胞，之后使用PureLinkTMRNA Mini Kit(Thermofisher)提取RNA。提取总RNA使用novaseq 6000平台进行RNA-seq测序。获得的测序结果通过STAR软件比对到人基因组，然后通过StringTie软件计算基因表达量。与对照组相比，发现Cas13X1、Cas13g3和RfxCas13d对应的脱靶位点对应的脱靶位点分别为102、133和323。表明Cas13g3的特异性与Cas13X1相当，而其脱靶效应则低于RfxCas13d(图17)。图17中KD代表敲低效率，DG代表下调基因数目。
实施例7：探究Cas13反式切割活性
细胞培养，转染
HEK293T细胞在含有10％FBS(Gibco)和1％Penicillin-Streptomycin(Gibco)的DMEM(Gibco)培养基中培养。使用0.25％Trypsin-EDTA(Gibco)消化细胞，转入24孔板中培养12小时，等细胞密度达到90％时，使用LIPOFECTAMINE 3000 Reagent(Invitrogen)进行转染。
细胞活性分析
细胞活性实验组每个孔转染600ng pCAG-Cas13-2A-eGFP质粒和300ng pUC19-U6-3'-DR crRNA质粒，crRNA靶向ANXA4 mRNA。对照组不转染任何质粒。使用Calcein-AM/PI double strain kit(Solarbio)对转染2天后的HEK293T进行荧光细胞活力测定。总细胞定量在490nm激发和515nm发射，死细胞定量在535nm激发和617nm发射。使用流式细胞仪进行测量。通过鉴定转染细胞活性，发现转染Cas13蛋白后，不影响细胞活性(图18A)，具体细胞活性信息见表9A。图18每组都有3个生物学复本，使用双尾t-检验评价统计学意义。
表9A：
反式切割活性
反式切割实验组每个孔转染600ng pCAG-Cas13-2A-eGFP质粒、10ng pCAG-eBFP-EF1a-mCherry质粒和300ng pUC19-U6-3'-DR crRNA质粒，其中crRNA靶向mCherry mRNA。反式切割对照组转染非靶向的U6-crRNA质粒。通过测量mCherry的荧光强度计算Cas13蛋白的顺式切割能力，同时通过测量EGFP的荧光强度计算反式切割能力。与之前的结果一致，Cas13X1、RfxCas13d和Cas13g3可以显著敲低mCherry mRNA(图18B)。与此同时，Cas13X.1和Cas13g3没有检测到反式切割活性，而RfxCas13d对EGFP转录本显示出显著的反式切割活性(图18B)。具体MFI结果信息见表9B。
表9B：

实施例8：高编辑效率的Cas13g3系统特征鉴定
细胞培养，转染
HEK293T细胞在含有10％FBS(Gibco)和1％Penicillin-Streptomycin(Gibco)的DMEM(Gibco)培养基中培养。使用0.25％Trypsin-EDTA(Gibco)消化细胞，转入24孔板中培养12小时，等细胞密度达到90％时，使用LIPOFECTAMINE 3000 Reagent(Invitrogen)进行转染。
Cas13g3系统最优spacer长度研究
为了探究Cas13g3系统的最优spacer长度，我们构建了靶向mCherry mRNA且spacer长度从5到50nt不等的U6-crRNA质粒。其中spacer长度在15-30nt之间，每隔1nt设计一条crRNA；当spacer长度为5-15nt和30-50nt时，每隔5nt设计一条crRNA。600ng pCAG-Cas13-2A-eGFP质粒、10ng pCAG-eBFP-EF1a-mCherry质粒和300ng pUC19-U6-3'-DR crRNA质粒三者共转到HEK293T细胞中作为实验组。600ng pCAG-Cas13-2A-eGFP质粒、300ng非靶向的U6-crRNA质粒和10ngpCAG-eBFP-EF1a-mCherry质粒三者共转到HEK293T细胞中作为对照组。实验组EGFP和mCherry双阳性细胞的MFI与阴性对照进行了标准化处理，标准化结果可以反映不同spacer长度对RNA干扰影响。结果显示，当使用长度为27nt的spacer时，Cas13g3在两个靶标位点的平均敲除效率最高(图19)。具体编辑结果信息见表10。
表10：

Cas13g3系统PFS偏好分析
为了分析Cas13g3系统的PFS偏好性，在EF-1α-mCherry质粒的mCherry基因上游克隆了16种PFS组合的目标。实验组EGFP和mCherry双阳性细胞MFI与阴性对照的MFI进行标准化，实验组为转染600ng pCAG-Cas13-2A-eGFP质粒、10ngpCAG-eBFP-EF1a-mCherry质粒和300ng pUC19-U6-3'-DR crRNA质粒的HEK293T细胞；阴性对照为转染了600ng pCAG-Cas13-2A-eGFP质粒、300ng非靶向的U6-crRNA质粒和10ngpCAG-eBFP-EF1a-mCherry质粒的HEK293T细胞。结果显示，Cas13g3蛋白在这16种PFS序列中都展示了稳定且高效的干扰效果(图20)。具体编辑结果信息见表11(其中的spacer序列为：SEQ ID NO.81:GCCGGCACTGATGAGGGCTGCCTAATT)。
表11：

NLS对Cas13g3系统干扰活性的影响
为了鉴定NLS和NES对Cas13g3系统干扰活性的影响，我们构建了3个分别融合NLS、融合NES和没有融合NLS和NES的Cas13蛋白表达质粒(图21)。600ng pCAG-Cas13-2A-eGFP质粒、10ng pCAG-eBFP-EF1a-mCherry质粒和300ng pUC19-U6-3'-DR crRNA质粒三者共转到HEK293T细胞中作为实验组。对照组只转染pCAG-eGFP质粒。分析并计算EGFP和mCherry双阳性细胞的MFI，与对照组的标准化结果显示相对于融合NES或者没有任何定位信号，NLS序列的融合可以提高Cas13g3的敲除效率(图21)。具体编辑结果信息见表12。
表12：
因此，最佳的Cas13g3编辑器组成为27nt的spacer长度的crRNA质粒和融合NLS序列的Cas13g3蛋白质粒。
实施例9：最优的Cas13g3编辑器干扰能力验证
细胞培养，转染
HEK293T细胞在含有10％FBS(Gibco)和1％Penicillin-Streptomycin(Gibco)的DMEM(Gibco)培养基中培养。使用0.25％Trypsin-EDTA(Gibco)消化细胞，转入24孔板中培养12小时，等细胞密度达到90％时，使用LIPOFECTAMINE 3000 Reagent(Invitrogen)进行转染。实验组每个孔转染600ng pCAG-Cas13-2A-eGFP质粒和300ng pUC19-U6-3'-DR crRNA质粒。非靶标对照组转染600ng pCAG-Cas13-2A-eGFP质粒和300ng非靶标的U6-crRNA质粒。
RNA提取，RNA定量分析
转染48小时后使用Reagent(Life Technologie)裂解细胞，之后使用PureLinkTMRNA Mini Kit(Thermofisher)提取RNA。最后通过II One Step qRT-PCR SYBR Green Kit(Vazyme)进行RNA定量分析。RT-qPCR结果采用2-ΔΔCT法进行分析，利用靶基因与内参基因GAPDH在3个生物学复本中的平均CT值的差异计算靶基因的相对表达量，并以对照组的相对表达量进行标准化。
Cas13g3系统与其他Cas13系统RNA干扰能力的比较
为了系统性地比较Cas13g3蛋白与目前最常用且切割效率最高的Cas13蛋白Cas13X1和RfxCas13d在相同靶标位点的RNA干扰活性并且更大规模地研究Cas13g3蛋白的敲除效率，我们设计了靶向5个内源基因的一共15个crRNAs质粒。通过靶向5个体内基因EZH2、NF2、HRAS、NRAS和PPARG，Cas13X1、RfxCas13d和Cas13g3能够在EZH2、NF2、HRAS、NRAS和PPARG基因上展现强大的RNA干扰活性，它们的平均敲低效率分别为52.84％、54.58％和60.37％(图22)，具体编辑结果见表13。干扰结果进行配对t检验(paired-t-test)统计学分析显示Cas13g3具有高效的RNA敲低活性，与Cas13X1和RfxCas13d相当。
表13：
尽管以上结合附图对本发明的实施方案进行了描述，但本发明并不局限于上述的具体实施方案和应用领域，上述的具体实施方案仅仅是示意性的、指导性的，而不是限制性的。本领域的普通技术人员在本说明书的启示下和在不脱离本发明权利要求所保护的范围的情况下，还可以做出很多种的形式，这些均属于本发明保护之列。

Claims (15)

  1. 经分离的Cas13核酸酶蛋白，所述Cas13核酸酶蛋白的氨基酸序列为：
    a)如SEQ ID NO.1～28中任一序列所示的氨基酸序列；或
    b)与SEQ ID NO.1～28中的任一序列具有至少80％的序列同一性、且具有RNA切割活性的氨基酸序列。
  2. 如权利要求1所述的Cas13核酸酶蛋白，其中所述Cas13核酸酶蛋白是Cas13bt1、Cas13bt2、Cas13g、Cas13h、Cas13i、Cas13j或Cas13k蛋白，优选为Cas13g3。
  3. 一种工程化的Cas13核酸酶效应蛋白，包含Cas13核酸酶蛋白，该Cas13核酸酶蛋白包含:
    a)如SEQ ID NO.1～28中任一序列所示的氨基酸序列；或，
    b)与SEQ ID NO.1～28中的任一序列具有80％以上序列同一性、且具有RNA切割活性的氨基酸序列。
  4. 如权利要求3的所述效应蛋白，其中所述Cas13核酸酶蛋白通过氨基酸突变失去催化活性，例如所述Cas13核酸酶蛋白在其C端和/或N端的HEPN结构域(RxxxxH基序)通过氨基酸突变形成dCas13蛋白。
  5. 权利要求3或4所述的效应蛋白，其还包含与所述Cas13核酸酶蛋白融合的功能结构域。
  6. 如权利要求5所述的效应蛋白，所述功能结构域选自如下的一项或多项：翻译起始结构域、翻译阻遏结构域、反式激活结构域、表观遗传修饰结构域、核碱基编辑结构域、逆转录酶结构域、报告分子结构域和核酸酶结构域。
  7. 如权利要求6所述的效应蛋白，其中所述核碱基编辑结构域为腺苷脱氨酶、胞苷脱氨酶或它们的催化结构域。
  8. 一种多核苷酸，其编码权利要求1或2的Cas13核酸酶蛋白或权利要求3-7任一项的效应蛋白。
  9. 包含如权利要求8所述的多核苷酸的载体，所述载体为质粒或病毒，所述病毒优选为慢病毒。
  10. 一种工程化的CRISPR-Cas13基因编辑系统，包含：
    (a)如权利要求1或2所述的核酸酶、如权利要求3-7中任一项所述的工程化Cas13核酸酶效应蛋白或编码所述效应蛋白的核酸；以及
    (b)crRNA，其包含与靶核酸中的靶序列互补的间隔区序列；
    其中所述工程化的Cas13核酸酶效应蛋白和所述crRNA能够形成CRISPR复合物，所述CRISPR复合物特异性结合包含所述靶序列的靶核酸并诱导所述靶核酸的修饰。
  11. 如权利要求10所述的CRISPR-Cas13基因编辑系统，其中所述crRNA还包含直接重复(DR)序列，优选所述直接重复(DR)序列包含SEQ ID NO.29～56中任一序列所示的序列，或包含与SEQ ID NO.29～56中任一序列所示的序列具有至少80％同一性的序列。
  12. 包含如权利要求10或11所述的工程化的CRISPR-Cas13基因编辑系统的试剂盒。
  13. 如权利要求1或2所述的Cas13核酸酶蛋白、如权利要求3-7中任一项所述的工程化的CRISPR-Cas13核酸酶效应蛋白、如权利要求10或11所述的工程化的CRISPR-Cas13基因编辑系统在制备治疗与个体的细胞中与核酸突变相关的疾病或病症的药物中的用途。
  14. 一种修饰细胞中包含靶核酸的方法，包括使所述细胞与如权利要求1或2所述的Cas13核酸酶蛋白、如权利要求3-7任一项所述的工程化的 Cas13核酸酶效应蛋白、如权利要求10或11所述的工程化的CRISPR-Cas13基因编辑系统接触，从而实现对所述细胞中所述靶核酸的修饰。
  15. 包含如权利要求1或2所述的Cas13核酸酶蛋白、如权利要求3-7任一项所述的工程化的Cas13核酸酶效应蛋白或如权利要求10或11所述的工程化的CRISPR-Cas13基因编辑系统的组合物，其用于核酸的修饰。
PCT/CN2023/101348 2022-04-26 2023-06-20 经分离的Cas13蛋白、基于它的基因编辑系统及其用途 Ceased WO2023208256A1 (zh)

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