CN112430586B

CN112430586B - VI-B type CRISPR/Cas13 gene editing system and application thereof

Info

Publication number: CN112430586B
Application number: CN202011280752.XA
Authority: CN
Inventors: 胡争; 崔资凤; 黄昭玥; 李利芳
Original assignee: Zhuhai Shutong Medical Technology Co ltd
Current assignee: Zhuhai Shutong Medical Technology Co ltd
Priority date: 2020-11-16
Filing date: 2020-11-16
Publication date: 2021-09-07
Anticipated expiration: 2040-11-16
Also published as: CN112430586A

Abstract

The invention relates to the technical field of gene editing, in particular to a VI-B type CRISPR/Cas13 gene editing system and application thereof. The VI-B type CRISPR/Cas13 gene editing system comprises an Lt1Cas13B protein and CRISPR RNA; the Lt1Cas13b protein is an RNA endonuclease, and the Lt1Cas13b protein has an amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence which is at least 80% identical to the amino acid sequence shown in SEQ ID NO. 1. The invention provides a novel VI-B type CRISPR/Cas13 gene editing system, which can be widely applied to the scenes in which prokaryotes or eukaryotes RNA need to be identified, combined and edited and provides a new application choice for an RNA editing tool.

Description

VI-B type CRISPR/Cas13 gene editing system and application thereof

Technical Field

The invention relates to the technical field of gene editing, in particular to a VI-B type CRISPR/Cas13 gene editing system and application thereof.

Background

Gene editing (gene editing) technology makes it possible to modify DNA sequence sites, such as Zinc Finger Nucleases (ZFNs) as a first generation gene editing tool, transcription activator-like effectors (TALENs), CRISPRs (Clustered Regularly Interspaced Short Palindromic repeats) of type II and type V in a third generation gene editing tool, and Clustered regulated partitioned Short Palindromic Repeat/Cas (CRISPR-associated protein) can be used for targeted modification of genomes, but these gene editing systems can only target genomes and foreign DNA, but not RNA.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) type VI system is a natural immune system from archaea and bacteria. The target RNA sequence is identified by utilizing the nucleic acid base complementary pairing principle, the Cas effector protein is guided to carry out targeted cutting, and the target RNA sequence is high in applicability, simple in design and high in efficiency. The VI-B type CRISPR/Cas13 gene editing system is a VI type CRISPR/Cas system which is widely applied at present. This system allows targeted editing of single-stranded RNA in prokaryotes by recognition and cleavage of the PFS (pro-spacer annealing site) sequence flanking the pro-spacer on both sides of the targeted polynucleotide, whereas in eukaryotes the CRISPR/Cas system type VI-B does not have PFS sequences for targeted editing of RNA.

In large and diverse metagenomes, microorganisms that have not been cultured or even discovered are hidden, and there may be a large number of undiscovered CRISPR VI-B/Cas 13 systems whose activity in prokaryotes and eukaryotes, as well as in an in vitro environment, needs to be confirmed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a VI-B type CRISPR/Cas13 gene editing system and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that: provides a VI-B type CRISPR/Ca s13 gene editing system, which comprises Lt1Cas13B protein and CRISPR RNA;

the Lt1Cas13b protein is an RNA endonuclease, and the Lt1Cas13b protein has an amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence which is at least 80% identical to the amino acid sequence shown in SEQ ID NO. 1.

The VI-B type CRISPR/Cas13 gene editing system has a functional structure comprising a crRNA secondary structure, a Cas13B effector protein functional domain or a Cas13B-crRNA complex structure.

The Lt1Cas13b protein comprises 986 amino acids and is a multi-domain and multifunctional RNA endonuclease. It cleaves single-stranded RNA complementary to sgRNA through two HEPN-like nuclease domains.

As a preferred embodiment of the VI-B type CRISPR/Cas13 gene editing system of the present invention, the RNA is prokaryotic or eukaryotic RNA. In prokaryotes, a variety of different PFS (promoter annealing site) sequences can be recognized, and the Lt1Cas13b protein cleaves single-stranded RNA complementary to sgRNA between PFS sequences through different nuclease domains. While acting in eukaryotes without PFS (promoter annealing site) sequences, the nuclease domain of Cas13b protein can cleave single-stranded RNA complementary to sgRNA independently of PFS sequences.

In prokaryotes, Cas13b is able to recognize multiple cleavage sites flanking a Protospacer (PFS) on both sides of an adjacent targeting sequence to form a single-stranded RNA molecule, and two important factors are required for recognition of the targeting sequence by the Cas13b protein: one is the nucleotide complementary to the crRNA spacer sequence, and the other is the flanking site (PFS) sequence of the original spacer sequence adjacent to the complementary sequence. However, in eukaryotes, the targeting editing action of Cas13b on RNA does not require pro-spacer flanking site (PFS) sequences. Therefore, the Protospacer sequence is designed according to the PFS selection of Cas13b reported in the literature, and the Cas13b is shown to have a cleavage effect in a prokaryotic system through interference experiments. Because PFS is not required for it to cleave RNA in eukaryotes, this CRISPR-Cas 13b system can target nearly all RNA sequences of interest in the eukaryotic genome by artificially designing the spacer sequence in the crRNA.

As a preferred embodiment of the type VI-B CRISPR/Cas13 gene editing system of the present invention, the CRISPR RNA is transcribed from a CRISPR Array comprising a spacer sequence and a direct repeat.

Since CRISPR RNA is produced by CRISPR Array transcription, CRISPR RNA also includes a spacer and direct repeat, and the CRISPR RNA sequence is, from 5-to 3-terminus: 5 '-spacer-direct repeat-3'.

CRISPR RNA (crRNA) in the invention guides Cas protein to recognize invading exogenous genome in the form of base complementary pairing. When the bacteria is exposed to bacteriophage or virus and the like for invasion, a short fragment of exogenous RNA is subjected to reverse transcription to form DNA and is integrated between CRISPR direct repetitive sequences in a host chromosome as a new spacer, so that genetic record of infection is provided, and when the organism is invaded by the exogenous gene again, CRISPR array is transcribed to generate precursor crRNA (pre-crRNA), the 5' end of the precursor crRNA is provided with the spacer sequence, the length of the precursor crRNA is 30nt, and the precursor crRNA is complementary with the sequence from the exogenous invasion gene; the 3' end is a repetitive sequence with the length of 36 nt. The pre-crRNA is subjected to Cas13 nuclease to remove 7nt upstream of the repetitive sequence to form mature crRNA. Mature crRNA, which contains an exogenous RNA sequence complementary to the spacer sequence, typically 23nt in length, serves as a guide RNA (sgrna) for Cas13 b.

As a preferred embodiment of the VI-B type CRISPR/Cas13 gene editing system, the length of the spacer sequence is 23-30 base sequences.

As a preferred embodiment of the VI-B type CRISPR/Cas13 gene editing system, the spacer sequence is shown as SEQ ID NO. 4.

As a preferred embodiment of the type VI-B CRISPR/Cas13 gene editing system of the present invention, the direct repeat sequence comprises 2 stem-loop structures.

As a preferred embodiment of the VI-B type CRISPR/Cas13 gene editing system, the direct repetitive sequence is a 36-base sequence.

As a preferred embodiment of the VI-B type CRISPR/Cas13 gene editing system, the direct repetitive sequence is shown as SEQ ID NO. 3.

As a preferred embodiment of the VI-B type CRISPR/Cas13 gene editing system, CRISPR RNA has an RNA sequence shown in SEQ ID NO. 2.

The invention also provides application of the VI-B type CRISPR/Cas13 gene editing system in recognizing, combining and editing prokaryotic or eukaryotic RNA.

As a preferred embodiment of the application of the invention, the specific operation of the application is as follows:

(1) transforming the expression vector for the Lt1Cas13b protein and the expression vector for CRISPR RNA into a cell;

(2) culturing and screening to obtain a transformant.

The invention has the beneficial effects that:

(1) according to the invention, a new VI-B type CRISPR/Cas13 gene editing system is excavated through the bioinformatics analysis of metagenome, and the gene editing system is applied to editing genes of prokaryotes or eukaryotes, so that a new choice is provided for a gene editing tool kit.

(2) The newly-discovered VI-B type CRISPR/Cas13 gene editing system provided by the invention has new physicochemical properties and can carry out targeted editing on single-stranded RNA.

Drawings

Fig. 1 is a composition diagram of the VI-B type CRISPR/Cas13B gene editing system of the present invention.

FIG. 2 is a diagram of prediction of RNA secondary structure of guide RNA molecule recognized by VI-B type CRISPR/Cas13B gene editing system.

FIG. 3 is a schematic diagram of an interference experiment of the VI-B type CRISPR/Cas13 gene editing system.

Fig. 4 is a schematic diagram of the results of determining the length of the direct repeat sequence of the VI-B type CRISPR/Cas13 gene editing system that functions at the prokaryotic level.

Fig. 5 is a schematic diagram of the results of determining the length of the spacer sequence that the VI-B type CRISPR/Cas13 gene editing system plays a role at the prokaryotic level.

FIG. 6 is a histogram of relative expression obtained by qPCR relative quantification after targeted cleavage of endogenous genes by the VI-B type CRISPR/Cas13 gene editing system of the invention.

Detailed Description

To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.

The sequences involved in the invention are:

sequence of	Numbering
		Lt1Cas13b protein amino acid sequence	SEQ ID NO:1
CRISPR RNA sequence	SEQ ID NO:2
		Direct repeat sequences of CRISPR Array	SEQ ID NO:3
Spacer sequences for CRISPR Array	SEQ ID NO:4

Example 1

This example analyzes and predicts the Lt1Cas13 b-related genes described in the present invention.

(1) Materials: metagenomic sequencing data.

(2) Software: fastqc (v0.11.5), fastp (v0.19.8), SOAPnuke (v1.5.2), hista2(v2.0.4), samtools (v1.9), iTools (v0.23), GeneMark (v3.38), IDBA-UD (v1.1.3), Bowtie2(v2.3.5.1), CD-HIT (v4.6), hummer (3.1b2), BLAST (v2.3.0+), Ordination (v1.0), WilcoxonTest (v1.0), environmentrAnalysis (v1.0)

(3) Detection method

The data source of the embodiment is metagenome sequencing data, and all possible gene sequences in the metagenome are obtained by performing quality control, splicing and gene prediction on the metagenome data.

The specific operation of the raw data of the metagenome is as follows:

(a) firstly, performing instruction control by using fastqc, checking whether data is original data or cleaned data, if the data is the original data, cleaning the original data and removing joints by using fastp, and if the data is the cleaned data, directly entering the next step;

(b) assembling the metagenome data by using various second-generation sequencing assembly software (such as IDBA-UD and SOAPdenovo2) to generate long fragment contigs;

(c) after removing the too short contigs, using MetaGeneMark in GeneMark software to carry out gene prediction, translating the obtained gene, and filtering out a sequence with less than 600 amino acids;

(d) carrying out homology clustering on protein sequences on the candidate sequences by using an OrthoFinder to generate candidate protein families, carrying out multi-sequence alignment on the protein family sequences, and determining the candidate protein families with a HEPN domain model (E … … RxxxxxxH); constructing the HMM model of the protein family, searching and comparing HMMERs again, and expanding the protein family;

(e) comparing the main stream database (nr, swiss prot) to obtain a gene which is not annotated as CRISPR-Cas system related protein;

(f) the contig sequence corresponding to the Cas protein was retrieved.

Example 2

In this embodiment, the obtained contigs possibly related to the type 2 CRISPR-Cas system are analyzed to obtain the predicted proteins and related elements related to the CRISPR-Cas system; namely, CRISPR sequence related to CRISPR/Cas13 and Cas13 protein prediction were performed on contigs obtained in example 1.

(1) Materials: contig sequence obtained in example 1, which may be related to CRISPR-Cas system.

(2) Software: CRISPRACASFinder (v4.2.19), python (v3.7.4), bedtools (v2.25.0).

(3) The detection method comprises the following steps:

the specific operation is as follows:

(a) analyzing and predicting the contig which is possibly related to the CRISPR (clustered regularly interspaced short palindromic repeats) Finder system 2 by using the CRISPR enveloping to obtain proteins and elements related to the CRISPR-Cas system, such as Cas protein, Spacer, direct repeat and the like;

(b) annotating candidate type VI protein family subcategories: collecting HMM files of Cas13a, Cas13b, Cas13c and Cas13d effector proteins, carrying out HMMER alignment on protein families, and classifying candidate VI type protein families into 4 subtypes and unknown types according to effector protein classification and whether Cas1, Cas2, csx27, csx28 and WYL auxiliary proteins exist;

(c) and (3) constructing a phylogenetic tree, and determining a novel VI type effector protein: a phylogenetic tree is constructed by all known Cas13 protein sequences and candidate type VI protein family sequences, and a branch family different from the existing Cas13 protein on the phylogenetic tree is determined as a novel type VI effector protein.

The CRISPR/Cas13 gene editing system disclosed by the invention is composed as shown in figure 1. As can be seen from fig. 1, the Lt1Cas13b gene editing system described in the present invention comprises the following components: endonuclease Cas13b gene, crRNA (including repeat and spacer sequences). The Cas13 gene comprises 986 amino acids; mature crRNA directs Cas13b protein cleavage to target sequence.

Example 3

In this example, the RNA secondary structure of the guide RNA molecule identified by the Cas13b gene editing system of the present invention is predicted, and the obtained RNA secondary structure is shown in fig. 2.

(1) Materials: a repeat sequence;

(2) software: NUPACK (http:// www.nupack.org/partition/new);

(3) the prediction method comprises the following steps: the RNA secondary structure shown in FIG. 2 was obtained by using NUPACK in-line applications to mimic the secondary structure of repeat RNA at 37 ℃ in vitro.

As shown in fig. 2, pre-crRNA forms mature crRNA under the action of Lt1Cas13 nuclease to form guide RNA (guide RNA), and guide RNA includes two stem-loop structures.

Example 4

This example determines the cleavage ability of the novel type VI-B CRISPR/Cas13 gene editing system of the present invention at the prokaryotic level, as well as the potential gene editing ability among eukaryotes, by interference experiments.

(1) Materials: examples 1-3 predicted CRISPR/Cas13 gene editing system-associated genes.

(2) The verification method comprises the following steps: in this embodiment, a prokaryotic verification system is constructed for the VI-B type CRISPR/Cas13 gene editing system newly found in the present invention, and the cleavage effect is verified, specifically, the operations are as follows:

(a) the VI-B type CRISPR/Cas13 gene editing system (comprising endonuclease Cas13B and CRISPR array) is inserted into a pET28a vector, the Lt1Cas13B protein is subjected to escherichia coli codon optimization, an artificially synthesized spacer sequence is added into the CRISPR array, and a strong heterologous promoter J23119 is added to the Lt1Cas13B protein and the CRISPR array to construct a prokaryotic expression pET28a-Cas13B plasmid;

(b) inserting an artificially synthesized target sequence corresponding to a CRISPR array spacer sequence after a first initiation codon of a chloramphenicol gene of a pACYC184 plasmid, and inserting a PFS sequence reported in a literature at the upstream and the downstream of the target sequence to construct a pACYC184-target plasmid;

(c) the plasmid containing pACYC184-target or no-load pACYC184 and pET28a-Cas13b are jointly transformed into Escherichia coli DH5a, the cells are diluted according to gradient after being recovered for 1h at 37 ℃, and the diluted cells are dripped on SOB culture medium containing kanamycin (50ug/ml) and chloramphenicol (30ug/ml) double resistance to be incubated for 12-16h at 37 ℃, and the colony number of single clone under different concentration gradient is observed.

The results of the interference experiments are shown in fig. 3, and the results show that, in combination with the PFS sequence reported in the literature, Cas13b can effectively target-cleave the RNA sequence in escherichia coli through the interference experiments. The right column of FIG. 3 is the target plasmid for targeted cleavage of Lt1Cas13b, the left column of FIG. 3 is the single Lt1Cas13b, and the number of single colony observed by gradient dilution is obviously reduced, which shows that the number of colony in the right column is obviously reduced, thus the Lt1Cas13b can effectively target and cleave the RNA sequence in Escherichia coli.

Example 5

This example determines the length of the direct repeat sequence of the VI-B type CRISPR/Cas13 gene editing system of the present invention acting at the prokaryotic level by interference experiments.

Materials: examples 1-3 predicted CRISPR/Cas13 gene editing system-associated genes.

The verification method comprises the following steps: in this embodiment, prokaryotic verification systems with direct repetitive sequences of 36bp, 33bp, 30bp, 25bp and 20bp are constructed for the VI-B type CRISPR/Cas13 gene editing system of the present invention, and the cleavage effect is verified, specifically, the following operations are performed:

(a) the VI-B type CRISPR/Cas13 gene editing system (comprising endonuclease Cas13B and CRISPR array) is inserted into a pET28a vector, the Cas13B protein is subjected to codon optimization of escherichia coli, direct repetitive sequences in the CRISPR array are gradually shortened, prokaryotic verification systems with the direct repetitive sequences of 36bp, 33bp, 30bp, 25bp and 20bp are constructed, artificially synthesized spacer sequences are added into the CRISPR array, and a strong heterologous promoter J23119 is added to an Lt1Cas13B protein sequence and the CRISPR array, so that a plasmid is constructed: pet28a-Cas13b-36DR, pet28a-Cas13b-33DR, pet28a-Cas13b-30DR, pet28a-Cas13b-25DR, pet28a-Cas13b-20 DR;

(b) inserting an artificially synthesized target sequence corresponding to a CRISPR array spacer sequence after a first start codon of a chloramphenicol gene of a pACYC184 plasmid to construct a pACYC184-target plasmid;

(c) the pET28a-Cas13b plasmids (pET28a-Cas13b-36DR, pET28a-Cas13b-33DR, pET28a-Cas13b-30DR, pET28a-Cas13b-25DR, pET28a-Cas13b-20DR) and pACYC184-target are jointly electroporated into Escherichia coli DH5a, the Escherichia coli DH5a is revived at 37 ℃ for 1h, the bacterial liquid is diluted in gradient, and the bacterial liquid is dripped on SOB culture medium containing kanamycin (50ug/ml) and chloramphenicol (30ug/ml) double resistance to incubate at 37 ℃ for 12-16h, and the number of single colony under different concentration gradient is observed.

The results are shown in fig. 4, where Lt1Cas13b effectively targets the DR sequence length of crRNA required for RNA cleavage to be 36.

Example 6

This example determines the length of the spacer sequence that the novel type VI-B CRISPR/Cas13 gene editing system of the present invention functions at the prokaryotic level by interference experiments.

(2) The verification method comprises the following steps: in this embodiment, the VI-B type CRISPR/Cas13 gene editing system constructed spacer sequences newly found in the present invention are 23bp, 22bp, 21bp, and 20bp, respectively, the direct repeat sequence is set to 36bp according to the conclusion obtained in embodiment 5 and the result of sequencing by combining small RNA, and the cleavage effect is verified in a prokaryotic verification system, and the specific operations are as follows:

(a) the VI-B type CRISPR/Cas13 gene editing system (comprising endonuclease Cas13B and CRISPR array) newly discovered by the invention is inserted into a pET28a vector, Lt1Cas13B protein is used for carrying out escherichia coli codon optimization, a direct repetitive sequence is set to be 36bp, a spacer sequence in the CRISPR array is gradually shortened, prokaryotic verification systems with the spacer sequences respectively being 23bp, 22bp, 21bp and 20bp are constructed, a strong heterologous promoter J23119 is added on the Lt1Cas13B protein and the CRISPR array, and a prokaryotic expression pET28a-C as13B plasmid is constructed: pet28a-Cas13b-36DR-23spacer, pet28a-Cas13b-36DR-22spacer, pet28a-Cas13b-36DR-21spacer, pet28a-Cas13b-36DR-20 spacer;

(c) the pET28a-Cas13b plasmids (pET28a-Cas13b-36DR-23spacer, pET28a-Cas13b-36DR-22spacer, pET28a-Cas13b-36DR-21spacer, pET28a-Cas13b-36DR-20spacer) and pACYC184-target were co-electroporated into Escherichia coli DH5a, after 1h of recovery at 37 ℃, the bacterial solution was diluted in gradient and dropped on SOB medium containing kanamycin (50ug/ml) and chloramphenicol (30ug/ml) dual resistance, and incubated at 37 ℃ for 12-16h, and the number of single colony under different concentration gradients was observed.

The results are shown in FIG. 5: the spacer length of the Lt1Cas13b for efficient targeting of crRNA required for RNA cleavage was at least 23nt, thus giving a spacer length in the range of 23-30 nt.

Example 7

This example determines the cleavage ability of the VI-B type CRISPR/Cas13 gene editing system of the present invention at the eukaryotic nuclear level by targeting eukaryotic endogenous gene ANXA4 and a qPCR relative quantification method.

(2) The verification method comprises the following steps: in this embodiment, a eukaryotic verification system is constructed for the VI-B type CRISPR/Cas13 gene editing system, and the cleavage effect is verified, specifically operating as follows:

(a) the crRNA targeting the endogenous gene ANXA4 was designed according to the following principle, two target crRNA-1 and crRNA-2 were designed on the ANXA4 exon, and the length of the spacer (spacer) was 23 nt:

1) crRNA includes a spacer sequence (spacer) and a Direct Repeat sequence (DR), which is from 5 to 3: 5 '-spacer-direct repeat-3'.

2) The length of the spacer sequence of the crRNA is 23-30 base sequences;

3) the spacer sequence of the crRNA is reverse complementary to the sense strand of the ANXA4 gene;

4) the direct repeat sequence of crRNA is a 36 base sequence;

5) the direct repeat sequence of crRNA should contain 2 stem loop (stem loop) structures;

6) in the middle of the spacer of crRNA is a seed region, and no mismatch can occur when binding to the target sequence.

(b) The minircle vector (minircle vector is described in [1] Darqet AM, Rangara R, Kreiss P, Schwartz B, Naimi S, Delaure P, Crouzet J, Scherman D.Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer. Gene. Therr.1999 Feb; 6(2) 209-18.doi: 10.1038/sj.3300816. PMID:10435105 [2] CheZY, He CY, Eharrdt A, Kay MA. minor DNA vector of ultimate DNA respuls in and high-expression vector ex vivo; the invention is described in [1] Darqet AM, Rangaret M J.12 ] PMI AM, Pro, DNA, PCR 2] plasmid, DNA, PCR 2. 12. Biogene, Lipase, P. 15-2. TM. Probe, P.76. 12, P.11. Probe, P.76. 12, P.11. 12. Biogene, Probe, DNA, No. 11. 12, 2. 12. Pat.11. No.: 4152. 12. insert of nucleic acid, DNA, 2. 12. for example No. 11. for example No. 12. for inserting nucleic acid, DNA, 2. for example No. 2. 12. for example, 2. for gene, DNA, 2. 12. for gene, DNA, 2. 12. for example, DNA, 2. 12. for example, 2. 12. for example, DNA, 2. for example, DNA Dicer Lt1Cas13b, CRISPR array), Lt1Cas13b protein for human source optimization, and inserted into the above designed synthetic crRNA sequence. Adding a promoter EF-1 alpha on an Lt1Cas13b protein source, adding a human promoter U6 on a crRNA source, and constructing eukaryotic expression minicircle-Cas13b-crRNA plasmids which comprise a minicircle-Cas13b-crRNA-1 plasmid, a minicircle-Cas13b-crRNA-2 plasmid and a plasmid which does not target any endogenous gene, wherein a spacer sequence (spacer) targets the endogenous gene ANXA 4;

(c) transferring the constructed minicircle-Cas13b-crRNA plasmid into blank HEK293 cells in an Hp transfection mode, taking minicircle-Cas13b-crRNA-1 and minicircle-Cas13b-crRNA-2 plasmids as experimental groups, taking minicircle-Cas13b-crRNA-NT plasmids as negative control groups, taking a group of HEK293 cells without plasmids as blank control groups, culturing for 48h, collecting cells, extracting RNA, performing reverse transcription to obtain cDNA, performing qPCR relative quantitative analysis by taking GAPDH as an internal reference gene, and observing whether the expression quantity of a target endogenous gene ANXA4 of the experimental groups is reduced relative to the blank control group.

The relative quantification result of qPCR is shown in fig. 6, and the results indicate that the expression level of the target endogenous gene ANXA4 in the experimental group is decreased relative to the blank control group. The VI-B type CRISPR/Cas13 gene editing system has targeting cutting effect in eukaryotes.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Zhuhaishutong medical science and technology Limited

<120> VI-B type CRISPR/Cas13 gene editing system and application thereof

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Asn Asn Leu Ala Ala Tyr Asn Ser Glu Gln Thr Leu Leu Glu Lys Gln

805 810 815

Ile Arg Met Ser Ser Asn Phe Lys Leu Ala Lys Glu Asp Phe Arg Tyr

820 825 830

Leu Asp Phe Asp Arg Asp Phe Glu Ile Lys Ile Glu Gly Tyr Arg Lys

835 840 845

Ile Lys Asp Phe Gly Ile Phe Arg His Leu Ile Lys Asp Arg Arg Val

850 855 860

Pro Ser Ile Leu Ala Phe Tyr Thr Lys Tyr Lys Lys Leu Gly Ser Ile

865 870 875 880

Asn Glu Glu Ile Ile Arg Asn Glu Ile Leu Asp Phe Glu Tyr Tyr Lys

885 890 895

Ile Ser Ile Leu Lys Arg Val Leu Glu Ile Asp Lys Gln Ile Tyr Asn

900 905 910

Asn Leu Ile Gly Lys Asn Ile Ser Ile His Glu Lys Phe Ser Glu Asn

915 920 925

Val Asn Lys Leu Tyr Leu Glu Asn Gln Lys Leu Ala Asn Lys Ile Ile

930 935 940

Glu Ile Arg Asn Lys Leu Leu His Asn Lys Val Pro Glu Ile Asn Leu

945 950 955 960

Glu Asn Ile Gln Lys Thr Phe Ser Glu Thr Leu Phe Leu Glu Met Glu

965 970 975

Lys Ser Cys Asn Glu Leu Glu Arg Leu Ile

980 985

<210> 2

<211> 63

<212> RNA

<213> Artificial Synthesis

<400> 2

guugugaaug ccauauuuuu gaaagguaga aacaacgaag uuugcagcug gauacgacag 60

acg 63

<210> 3

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 3

gttgtgaatg ccatattttt gaaaggtaga aacaac 36

<210> 4

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 4

gaagtttgca gctggatacg acagacg 27

Claims

1. A VI-B type CRISPR/Cas13 gene editing system comprising an Lt1Cas13B protein and CRISPR RNA;

the Lt1Cas13b protein is an RNA endonuclease, and the amino acid sequence of the RNA endonuclease is shown as SEQ ID NO. 1; the sequence of CRISPR RNA is shown in SEQ ID NO. 2.

2. The type VI-B CRISPR/Cas13 gene editing system according to claim 1, wherein the CRISPR RNA is transcribed from a CRISPR Array comprising a spacer sequence and a direct repeat.

3. The type VI-B CRISPR/Cas13 gene editing system according to claim 2, wherein the spacer sequence is 23-30 base sequences in length.

4. The type VI-B CRISPR/Cas13 gene editing system according to claim 3, wherein the spacer sequence is shown as SEQ ID NO. 4.

5. The type VI-B CRISPR/Cas13 gene editing system according to claim 2, characterized in that the direct repeat sequence comprises 2 stem-loop structures.

6. The type VI-B CRISPR/Cas13 gene editing system according to claim 2, characterized in that the direct repeat sequence is a 36 base sequence.

7. The type VI-B CRISPR/Cas13 gene editing system according to claim 2, wherein the direct repeat sequence is shown as SEQ ID NO 3.

8. Use of the type VI-B CRISPR/Cas13 gene editing system of any of claims 1-7 for the recognition, binding and editing of prokaryotic or eukaryotic RNA.

9. The application according to claim 8, wherein the specific operation of the application is as follows:

(2) culturing and screening to obtain a transformant.