CN114075559A - Type 2 CRISPR/Cas9 gene editing system and application thereof - Google Patents

Abstract

The invention relates to a type 2 CRISPR/Cas9 gene editing system, and belongs to the technical field of gene editing. The gene editing system comprises a Cas9 protein, an accessory protein, CRISPR RNA and transactivation CRISPR RNA; the Cas9 protein is a DNA endonuclease, and the Cas9 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 excavates a type 2 CRSIPR/Cas9 gene editing system in Faecalibacculumdenum through bioinformatics analysis, and the gene editing system is applied to editing prokaryotic or eukaryotic genes and provides a new choice for a gene editing tool kit.

Description

Type 2 CRISPR/Cas9 gene editing system and application thereof

Technical Field

The invention relates to a 2-type CRISPR/Cas9 gene editing system derived from Faecalibacculumdenum, belonging to the technical field of gene editing.

Background

Gene editing (gene editing) technology makes it possible to modify DNA sequence sites, for example, Zinc Finger Nucleases (ZFNs) which are first generation gene editing tools, and transcription-activated small nucleases (TALENs) which are similar to second generation gene editing tools can be used for modifying targeted genomes, but these methods are difficult to design, difficult to manufacture, expensive in cost and not strong in universality.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) system is a natural immune system from archaea and bacteria, and is a third generation gene editing tool. The method is different from the conventional gene editing tool (protein-DNA recognition), utilizes the nucleic acid base complementary pairing principle to recognize the target DNA sequence and guide the Cas effector protein to perform site-specific cleavage, and has the advantages of strong applicability, simple design, low cost and high efficiency. Cas proteins contain a variety of different effector domains (domains) that play a role in different activities such as nucleic acid recognition, stabilizing complex structures, hydrolyzing DNA phosphodiester bonds, and the like. Among them, the type II CRISPR/Cas9 system derived from Streptococcus pyogenes Cas (SpCas 9) is the most widely used CRISPR/Cas system due to its high cleavage efficiency. This system leaves a blunt-ended overhang and affects gene editing by identifying and cleaving the Protospacer Adjacent Module (PAM) sequence, i.e., "NGG," on the targeted polynucleotide.

In the large and diverse metagenome, uncultured or even undiscovered microorganisms are hidden, and there may be a large number of undiscovered CRISPR/Cas9 systems whose activity in prokaryotes and eukaryotes, as well as in an in vitro environment, needs to be confirmed.

A new anaerobic strain ALO17 was isolated in 2015 by the team Dr. Byoung-Chan Kim from the feces of laboratory mice C57BL/6J and was found to be closely related to Holdemanella biformis DSM 3989T, Faecalicoccus spodophyllophorus ATCC 29734T, Faecalialeacetacylindroids ATCC 27803T, and Allobaculiscoricois DSM 13633T by phylogenetic analysis of the prokaryotic 16SrRNA gene sequence (sequence similarity 87.4, 87.3, 86.9and 86.9%, respectively); on the basis of multiple taxonomic evidences, the species is identified as a new genus of Erysipelothricaceae family and named Faecalibaculumrodentium gen. nov., sp. nov., and scientists in all countries of the world have studied aiming at the strain in the fields of intestinal microbial environment, high-fat diet, intestinal microbial environment and tumorigenesis in the last five years, but the species is not reported in the field of gene editing.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a 2-type CRISPR/Cas9 gene editing system derived from Faecalibacculumdenum, which has new physicochemical properties and can identify a plurality of different PAM (protospacer adjacent motif) sequences including NGTA and NATA.

In order to achieve the purpose, the invention adopts the technical scheme that:

a type 2 CRISPR/Cas9 gene editing system derived from Faecalibaculumrodentium, comprising a Cas9 protein, an accessory protein, a crRNA: CRISPR RNA and tracrRNA: transactivation CRISPR RNA in the form of a complex of Cas9 protein and guide RNA formed by the binding of crRNA, tracrRNA-FrCas 9 protein complex;

the Cas9 protein is a DNA endonuclease, and the Cas9 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 Cas9 protein cleaves double-stranded DNA complementary to sgRNA upstream of the PAM sequence through different nuclease domains; the different nuclease domains are HNH-like nuclease domains or RuvC-like nuclease domains.

Description of mutations at several key amino acid positions for a specific Cas9 protein: mutation of amino acid site E to a at 796 may be a nickase nuclease; mutation of amino acid site E to a at 796 may be a nickase nuclease; mutation of amino acid position 902N to a may be nickase nuclease; mutation of amino acid 1010, position H, to a, may be a nickase nuclease; mutation of amino acid position 1013D to a may be a nickase nuclease; mutation at both amino acid 796, E, and amino acid 1013, D, is Cas9 nuclease where a can become non-cleaving but retains binding ability.

The helper proteins include a Cas1 helper protein, a Cas2 helper protein, and a Csn2 helper protein;

the Cas1 helper protein has an amino acid sequence shown as SEQ ID NO. 2, or an amino acid sequence at least 80% similar to the amino acid sequence shown as SEQ ID NO. 2;

the Cas2 helper protein has an amino acid sequence shown as SEQ ID NO. 3, or an amino acid sequence at least 80% similar to the amino acid sequence shown as SEQ ID NO. 3;

the Csn2 accessory protein has an amino acid sequence shown in SEQ ID NO. 4 or an amino acid sequence which is at least 80% similar to the amino acid sequence shown in SEQ ID NO. 4.

CRISPR RNA is generated by CRISPRARRArray transcription, CRISPR RNA has an RNA sequence shown as SEQ ID NO. 5, or an RNA sequence at least 80% similar to the nucleic acid sequence of SEQ ID NO. 5; the CRISPR Array comprises a direct repetitive sequence and a spacer sequence, wherein the direct repetitive sequence has a nucleic acid sequence shown in SEQ ID NO. 6 or a nucleic acid sequence which is at least 80 percent similar to the nucleic acid sequence shown in SEQ ID NO. 6; the spacer sequence has the nucleic acid sequence shown in SEQ ID NO. 7 or a nucleic acid sequence at least 80% similar to the nucleic acid sequence of SEQ ID NO. 7.

The transactivation CRISPR RNA comprises a sequence complementary to the CRISPR RNA direct repeat sequence, and the transactivation CRISPR RNA has the nucleic acid sequence shown in SEQ ID No. 8, or a nucleic acid sequence at least 70% similar to the nucleic acid sequence shown in SEQ ID No. 8.

The framework of the guide RNA formed by combining the crRNA and the tracrRNA is composed of 7-24nt of a direct repeat sequence of the crRNA and a tracrRNA sequence with a corresponding length, the two parts can be fused by a linker such as 'GAAA', 'TGAA' and 'AAAC' to form an sgRNA framework, preferably, the 20nt direct repeat sequence and the 71 nttracrRNA sequence are fused by 'GAAA' to form the sgRNA framework shown in SEQ ID NO:9, and further, the following 5 are included based on the results of the preferable variants of the sgRNA framework:

the sgRNA skeleton formed by fusing the first 18-14nt of the direct repetitive sequence of the crRNA with the last 69-65nt of the tracrRNA through a linker sequence such as GAAA:

sgRNA backbone length 91 nt: 18nt direct repeat +69nttracrRNA, as shown in SEQ ID NO: 10;

sgRNA backbone length 89 nt: a 17nt direct repeat +68nttracrRNA as shown in SEQ ID NO: 11;

length of sgRNA backbone 87 nt: 16nt direct repeat +67nttracrRNA, as shown in SEQ ID NO: 12;

length of sgRNA backbone 85 nt: 15nt direct repeat +66nttracrRNA, shown in SEQ ID NO: 13;

sgRNA backbone length 83 nt: 14nt direct repeat +65nttracrRNA, shown in SEQ ID NO: 14;

the nucleic acid sequences shown above include, but are not limited to, nucleic acid sequences that are at least 70% similar to SEQ ID NOS 9-14.

The 2-type CRISPR/Cas9 gene editing system derived from Faecalibacculumdenum combines or cuts specific DNA in a biological process, the combination or cutting of the specific DNA is realized by complementary pairing and recognition of guide RNA and a specific DNA target spot, and the length range of the pairing and combining part is 20-23bp nucleotide length; the specific DNA is DNA of prokaryotes or eukaryotes.

The length range of the pairing binding part for complementary pairing recognition of the guide RNA and the specific DNA target point is 21bp, 22bp or 23bp, wherein the Cas9 protein complex is highly sensitive to 14bp base mismatch close to an original spacer adjacent module (PAM) and is a seed region.

The sequence of the adjacent module of the original spacer sequence required for the function of combining or cutting DNA is 5 '-NNTA-3' at the downstream of the guide RNA/sgRNA recognition sequence.

The invention also provides application of the 2-type CRISPR/Cas9 gene editing system derived from Faecalibacculumdenum in editing procaryotic or eucaryotic DNA.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention excavates a type 2 CRSIPR/Cas9 gene editing system in Faecalibacculumdenum through bioinformatics analysis, and the gene editing system is applied to editing prokaryotic or eukaryotic genes and provides a new choice for a gene editing tool kit.

(2) The 2-type CRISPR/Cas9 gene editing system derived from Faecalibacculumdenum provided by the invention has new physicochemical properties and can identify various PAM sequences, and the PAM specific sequence identified by the gene editing system is 5 '-NNTA-3' at the downstream of a guide RNA/sgRNA identification sequence.

(3) The type 2 CRISPR/Cas9 gene editing system derived from Faecalibacculumdenum provided by the invention has higher cutting efficiency than the most common SpCas9 at the same DNA site and lower off-target than the most common SpCas9, and is a safer and more effective gene editing tool.

Drawings

FIG. 1 is a composition diagram of the type 2 CRISPR/Cas9 gene editing system of Faecalibacculumdenum of the invention.

FIG. 2 is a schematic structural diagram of Cas9 protein of the type 2 CRISPR/Cas9 gene editing system of Faecalibacculumden of the invention.

FIG. 3 is an RNA secondary structure prediction diagram and an optimal sgRNA framework diagram of a guide RNA molecule recognized by the type 2 CRISPR/Cas9 gene editing system of Faecalibaculumrodentium of the present invention.

FIG. 4 is a prokaryotic PAM sequence diagram of the type 2 CRISPR/Cas9 gene editing system of Faecalibacculumden according to the present invention.

FIG. 5 is a schematic diagram of a prokaryotic interference experiment of the Faecalibacculumden type 2 CRISPR/Cas9 gene editing system.

FIG. 6 is a schematic diagram of eukaryotic cleavage of the type 2 CRISPR/Cas9 gene editing system of Faecalibacculumdenum of the invention.

FIG. 7 is a schematic diagram of the optimal length of sgRNA framework of the Faecalibacculumdenum type 2 CRISPR/Cas9 gene editing system according to the invention.

FIG. 8 is a schematic diagram showing the optimal length of sgRNA recognition sequence of the type 2 CRISPR/Cas9 gene editing system of Faecalibacculumdenum of the invention.

FIG. 9 is a schematic diagram showing the comparison between the targeting efficiency and off-target of GUIDE-seq of the Faecalibaculumrodentium type 2 CRISPR/Cas9 gene editing system and SpCas 9.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

A type 2 CRISPR/Cas9 gene editing system derived from Faecalibaculumrodentium comprising a Cas9 protein, an accessory protein, CRISPR RNA and transactivation CRISPR RNA as shown in figure 1; the Cas9 protein is a DNA endonuclease, and the Cas9 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.

As a preferred embodiment of the type 2 CRISPR/Cas9 gene editing system derived from faecalibacculumn, the Cas9 protein cleaves double-stranded DNA complementary to sgRNA upstream of the PAM sequence through different nuclease domains, as shown in fig. 2; the different nuclease domains are HNH-like nuclease domains or RuvC-like nuclease domains.

The Cas9 protein (Faecalibacculumrodentium Cas9), abbreviated as FrCas9 protein, contains 1372 amino acids, and is a multi-domain and multifunctional DNA endonuclease. It efficiently cleaves double-stranded DNA complementary to sgRNA upstream of PAM through different nuclease domains, such as: DNA strands complementary to the sgRNA sequence are cleaved by HNH-like nuclease domain, or non-complementary strand DNA is cleaved by a RuvC-like nuclease domain. Wherein, the 796 amino acid site E mutation to A is a nickase nuclease; mutation of amino acid site E to a at 796 may be a nickase nuclease; mutation of amino acid position 902N to a may be nickase nuclease; mutation of amino acid 1010, position H, to a, may be a nickase nuclease; mutation of amino acid position 1013D to a may be a nickase nuclease; mutation at both amino acid 796, E, and amino acid 1013, D, is Cas9 nuclease where a can become non-cleaving but retains binding ability.

As a preferred embodiment of the inventive type 2 CRISPR/Cas9 gene editing system derived from faecalibacculumn rodentium, the auxiliary proteins include a Cas1 auxiliary protein, a Cas2 auxiliary protein and a Csn2 auxiliary protein; the Cas1 auxiliary protein has an amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence which is at least 80 percent identical to the amino acid sequence shown in SEQ ID NO. 2; the Cas2 helper protein has an amino acid sequence shown as SEQ ID NO. 3, or an amino acid sequence which is at least 80% identical to the amino acid sequence shown as SEQ ID NO. 3; the Csn2 accessory protein has an amino acid sequence shown in SEQ ID NO. 4, or an amino acid sequence which is at least 80% identical to the amino acid sequence shown in SEQ ID NO. 4.

The auxiliary proteins Cas1, Cas2 and Csn2 are involved in foreign gene capture and maturation of crRNA.

As a preferred embodiment of the type 2 CRISPR/Cas9 gene editing system derived from Faecalibacculumn, CRISPR RNA is transcribed by CRISPR Array, CRISPR RNA has an RNA sequence shown in SEQ ID NO. 5; the CRISPR Array comprises a direct repetitive sequence and a spacer sequence, wherein the direct repetitive sequence is shown as SEQ ID NO. 6, and the spacer sequence is shown as SEQ ID NO. 7.

CRISPR RNA (crRNA) in the invention guides Cas protein to recognize invading exogenous genome in a base complementary mode. When the bacteria are exposed to bacteriophage or virus and the like for invasion, short fragments of exogenous DNA are integrated between CRISPR repeated spacer sequences in a host chromosome as new spacers, thereby providing genetic record of infection, when the organisms are invaded by exogenous genes again, CRISPR array is transcribed to generate precursor crRNA (pre-crRNA), the 5' end of the precursor crRNA is spacer sequence, the length of the precursor crRNA is 30bp, and the precursor crRNA is complementary with the sequence from the exogenous invasion genes; the 3' end is a repetitive sequence with the length of 36 bp.

As a preferred embodiment of the type 2 CRISPR/Cas9 gene editing system derived from Faecalibacculumn rodentium, the transactivation CRISPR RNA comprises a sequence complementary to CRISPR RNA direct repeat sequence, and the transactivation CRISPR RNA is shown as SEQ ID NO. 8.

The trans-activated crRNA (tracrrna) is a non-protein coding RNA, and is involved in the maturation of crRNA and the formation of sgRNA. Under the action of the tracrRNA and Cas9 nuclease, pre-crRNA is removed from the upstream 0-16nt of the spacer sequence and the downstream 12-29nt of the repetitive sequence to form mature crRNA, and combined with the tracrRNA to form a tracrRNA-crRNA complex which comprises a part for recognizing exogenous DNA sequence and has the length ranging from 14-30 bp. The crRNA and crRNA can be ligated to form an sgRNA comprising two bull and three duplex structures upstream and one stem loop structure downstream by adding a four base tetra loop (e.g., "GAAA", "TGAA" or "AAAC" sequence) between downstream of the crRNA and upstream of the crRNA, and further divided into a portion that recognizes the foreign DNA sequence and a backbone portion.

The cutting action of the endonuclease can be further optimized by adjusting the length of the sgRNA for recognizing the part of the exogenous DNA sequence and the length of the tracrRNA. The previous experiments of the invention prove that the optimal length of the sgRNA for identifying the exogenous DNA sequence part is 21bp, 22bp or 23bp (as shown in figure 8), and the optimal length of the framework part is 5 as follows, as shown in figures 3 and 7: 91nt (18nt crRNA direct repeat +69nttracrRNA), 89nt (17nt crRNA direct repeat +68nttracrRNA), 87nt (16nt crRNA direct repeat +67nttracrRNA), 85nt (15nt crRNA direct repeat +66nttracrRNA), and 83nt (14 nt crRNA direct repeat +65 nttracrRNA);

as a preferred embodiment of the type 2 CRISPR/Cas9 gene editing system derived from Faecalibacculumn, the type 2 CRISPR/Cas9 gene editing system derived from Faecalibacculumn binds or cuts the structure of DNA function in the biological process.

As a preferred embodiment of the 2 type CRISPR/Cas9 gene editing system derived from Faecalibacculumn rodentium, the DNA is prokaryotic or eukaryotic DNA.

The FrCas9 is capable of recognizing a break (DSB) in which a DNA double-stranded molecule is formed by a variety of Protospacer Adjacent Modules (PAMs) immediately downstream of the targeting sequence, and two important factors are required for recognition of the targeting sequence by the FrCas9 protein: one is the nucleotide complementary to the crRNA spacer sequence, and the other is the Protospacer Adjacent Module (PAM) sequence Adjacent to the complementary sequence. The FrCas9 has a cutting effect in a prokaryotic system through a depletion experiment, and the positions 3 and 4 of the PAM sequence recognized by the newly discovered type 2 CRISPR/Cas9 system are preliminarily verified to be TA, as shown in FIG. 4. Interference experiments prove that PAM downstream of the targeting sequence recognized by the FrCas9 is 5 '-NNTA-3', as shown in FIG. 5, and the PAM is verified in eukaryotic experiments, as shown in FIG. 6. By artificially designing the spacer sequence in the crRNA, this CRISPR-Cas9 system can target almost all DNA sequences of interest in the genome, creating site-specific blunt-ended double-strand breaks (DSBs). (ii) the DSB is repaired by non-homologous ends, thereby generating small random insertions/deletions (indels) at the cleavage site to inactivate the gene of interest; alternatively, by high fidelity homologous repair, a homologous repair template can be used to make precise genomic modifications at the DSB site.

The invention also aims to provide application of the type 2 CRISPR/Cas9 gene editing system derived from Faecalibaccullumdentium in editing genes of prokaryotes or eukaryotes.

As a preferred embodiment of the application, the type 2 CRISPR/Cas9 gene editing system derived from Faecalibacculumden is used for combining or cutting the structure of DNA function at DNA level.

Example 1

In this embodiment, the RNA secondary structure of the guide RNA molecule recognized by the faecalibacculumn codentium type 2 CRISPR/Cas9 gene editing system is predicted, the RNA structure after the combination of the two is predicted by simulating the combination process of the tracrRNA and the RNA transcribed from repeat, and the obtained RNA secondary structure is shown in fig. 3.

(1) Materials: predicted tracrRNA and repeat sequences, and the predicted sequence of anti-repeat.

(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 on-line to simulate the interaction process of two RNAs at 37 ℃ in vitro, each in 1. mu.l, and then performing secondary structure prediction on the resulting RNA composition.

As shown in FIG. 3, the pre-crRNA is fused with the tracrRNA to form a tracrRNA-crRNA complex, which contains an exogenous DNA sequence complementary to the spacer sequence and has a length of 14-30bp, by removing 0-16nt upstream of the spacer sequence and 12-29nt downstream of the repeat sequence under the action of the tracrRNA and Cas9 nuclease. The sgrnas can be ligated together by adding a four base tetra loop between downstream of the crRNA and upstream of the tracrRNA to form a sgRNA comprising two bull and three duplex structures upstream and three stem loop structures downstream.

Example 2

In this embodiment, the primary spacer adjacent module (PAM) recognized by the faecalibacculumdenstitum type 2 CRISPR/Cas9 gene editing system in a prokaryotic system is 5 '-NNTA-3'.

(1) Materials: the CRISPR/Cas9 gene editing system related gene predicted by the implementation is obtained.

(2) The verification method comprises the following steps: in this embodiment, a prokaryotic verification system is constructed for the type 2 CRISPR/Cas9 gene editing system of Faecalibaculumrodentium described in the present invention, the cleavage effect of the prokaryotic verification system is verified, and the identified PAM sequence is primarily discovered by a second-generation sequencing technology, with the result shown in fig. 4;

the specific operation is as follows:

(a) inserting a 2-type CRISPR/Cas9 gene editing system (comprising endonuclease Cas9, auxiliary proteins Cas1, Cas2, Csn2, CRISPR array and non-coding RNA tracrRNA) of Faecalibaculumrodentium disclosed by the invention into a pACYC184 vector, carrying out Escherichia coli codon optimization on Cas9 protein, adding a natural spacer sequence and a spacer sequence in the crispR array, and adding a strong heterologous promoter J23119 on the Cas9 protein and the CRISPR array to construct a prokaryotic expression pACYC184-FrCas9 plasmid;

(b) adding 7 random bases (16384 insertion fragments in total) at the 3' position of the spacer sequence of the library, selecting two enzyme cutting sites of EcoRI and NcoI in the MCS multiple cloning of the pUC19 vector, cloning the library into the vector, and constructing a target-library plasmid;

(c) jointly transferring pACYC184-FrCas9 or unloaded pACYC184 plasmid and target-library into Escherichia coli DH5a, recovering for 2h at 25 ℃, uniformly smearing on SOB culture medium containing ampicillin sodium (100ug/ml) and chloramphenicol (34ug/ml) dual resistance, incubating for 30h at 25 ℃, and collecting the plasmid by alkaline lysis method;

(d) PCR amplification contains a spacer sequence and seven random bases, joint is added at two ends of a PCR product for second-generation sequencing, PAM exhaustion threshold (PPDV) relative to a no-load control group is calculated, and Weblogo is utilized to generate a PAM sequence of the 2 type CRISPR/Cas9 gene editing system of Faecalibaculumrodentium, wherein the PAM sequence is 5 '-NNTA-3'.

FIG. 3 is a schematic diagram of a conserved PAM sequence adjacent to a module of a primary spacer sequence identified by a Faecalibaculumrodentium type 2 CRISPR/Cas9 gene editing system in a prokaryotic system, secondary sequencing analysis is performed on library DNA obtained through a depletion experiment, a PAM depletion threshold (PPDV) relative to a no-load control group is calculated, and a PAM sequence generated from FrCas9 by Weblogo is 5 '-NNTA-3'.

Example 3

In this embodiment, an interference experiment is performed to verify that a Protospacer Adjacent Module (PAM) identified in the CRISPR/Cas9 type 2 gene editing system of Faecalibaculumrodentium described in the present invention determines the cleavage capability at the prokaryotic level and the potential gene editing capability in eukaryotes. The interference experiment result is shown in fig. 5, and a schematic diagram of a plurality of possible PAM sequences identified by the type 2 CRISPR/Cas9 gene editing system of Faecalibaculumrodentium is shown in fig. 5.

(1) Materials: pACYC184-FrCas9, target-library plasmid, PAM initially recognized, obtained in example 4.

(2) The verification method comprises the following steps: in this embodiment, an interference experiment is performed to further determine a primary spacer adjacent module (PAM) recognized by the type 2 CRISPR/Cas9 gene editing system of Faecalibaculumrodentium in a prokaryotic system;

the specific operation is as follows:

(a) adding 16 combination sequences of NNTA 3' of the spacer sequence, and cloning into pUC19 through EcoRI enzyme cutting sites and NcoI enzyme cutting sites to construct a target plasmid;

(b) 16 target plasmids are respectively transferred into an escherichia coli DH5a electrotransformation competence containing FrCas9 related gene loci, after recovery for 2h at 25 ℃, the plasmids are diluted in a gradient manner, and are incubated overnight at 25 ℃ on SOB culture medium containing ampicillin sodium (100ug/ml) and chloramphenicol (34ug/ml) double resistance by a dot coating method, and the number of monoclonal bacteria is observed.

FIG. 5 is a schematic diagram of an interference experiment of the Faecalibacculumdenum type 2 CRISPR/Cas9 gene editing system. On the basis of NNTA found in a depletion experiment, target plasmids of 16 NN different combinations are constructed, the number of single colony is observed through an interference experiment, the leftmost column of an A diagram in FIG. 5 is an independent FrCas9, the right side is a FrCas9 targeted cutting target plasmid, and the result shows that the number of colony in the right side column is reduced; FIG. 5, panel B, is a statistical graph of the cleavage effects of various PAM sequences recognized by the CRISPR/Cas9 gene editing system type 2 of Faecalibacculumrodentium of the present invention, which illustrates that cfu of target plasmids of 14 NN different combinations is decreased compared with that of the control group. The results show that the newly discovered CRISPR/Cas9 system is verified to recognize the original spacer adjacent module (PAM) as 5 '-NNTA-3' (N represents any base in A, T, C, G) in a prokaryotic system through interference experiments.

Example 4

In this embodiment, an interference experiment verifies that the tracrRNA range required for cutting a target DNA sequence is exerted in the type 2 CRISPR/Cas9 gene editing system of Faecalibaculumrodentium, and the result of the interference experiment is shown in a graph C in FIG. 5.

(1) Materials: pACYC184-FrCas9, target plasmid, primary identified PAM, obtained in example 5.

(2) The verification method comprises the following steps: in this embodiment, an interference experiment is performed to further determine that the type 2 CRISPR/Cas9 gene editing system of faecalibaccullumrodentium of the present invention exerts a tracrRNA range required for cutting a target DNA sequence in a prokaryotic system;

the specific operation is as follows:

(a) respectively cloning 6 gene sequences of all possible non-coding regions (Noncoding) in wild Faecalamustinum into a target plasmid by a Gibson cloning method to construct target-NC 1-6 plasmids, wherein NC6 is formed by splicing the full lengths of the non-coding regions, NC1-5 respectively represents the 1 st-5 th possible non-coding regions, a strong heterologous promoter J23119 is added at the upstream of each NC plasmid in the non-coding regions, and respectively represents the forward non-coding region by "+", and the "-" represents the reverse non-coding region;

(b) the pACYC184-FrCas9 obtained in example 5 is subjected to PCR homologous recombination to remove all possible non-coding regions, retain CRISPR-associated protein and CRISPR array gene sequences and construct pACYC 184-delta FrCas 9;

(c) 12 target-NC plasmids were transferred into E.coli DH5a electrotransformation competence containing pACYC 184-delta FrCas9, recovered at 25 ℃ for 2h, and then diluted in gradient, incubated overnight at 25 ℃ on SOB medium containing ampicillin sodium (100ug/ml) and chloramphenicol (34ug/ml) double resistance by dot coating, and the number of monoclonal bacteria was observed.

FIG. 5 is a schematic diagram of an interference experiment of the Faecalibacculumrodentium type 2 CRISPR/Cas9 gene editing system, and the result shows that the colony numbers of NC1 and 6 monoclonal are obviously less than those of NC 2-5, which indicates that the first alternative noncoding region can assist FrCas9 in effectively targeting and cutting a DNA sequence in Escherichia coli.

Example 5

In this embodiment, the ability of the Faecalibaculumrodentium type 2 CRISPR/Cas9 gene editing system to cut a target DNA sequence in a eukaryotic cell is verified through ODN experiments, the ODN-PCR result is shown in a diagram in fig. 6, and the Sanger sequencing result is shown in B diagram in fig. 6.

(1) Materials: all amino acid sequences, CRISPR array sequences, tracrRNA sequences and identified PAMs of the Faecalibaculumrodentium editing genes obtained in examples 1 to 6;

(2) the verification method comprises the following steps: in this embodiment, ODN experiments are performed to verify that the ability of the Faecalibacculumdenum type 2 CRISPR/Cas9 gene editing system of the invention to cut a target DNA sequence in eukaryotic cells,

the specific operation is as follows:

(a) synthesizing an adult-optimized FrCas9 protein sequence, cloning the protein sequence into a PX330 eukaryotic vector, and constructing a PX330-FrCas9 plasmid;

(b) selecting 30bp of NGTA, NATA, NCTA and NTTA upstream on a human RNF2 gene as a target sequence, constructing CRISPR array by a Gibson method, adding a human eukaryotic strong promoter U6 at the CRISPR array upstream, and constructing a PX330-FrCas9-array plasmid;

(c) adding a murine eukaryotic strong promoter U6 at the upstream of the tracrRNA determined in the example 4 to construct a PX330-FrCas9-array-tracrRNA plasmid;

(d) electrotransfering the PX330-FrCas9-array-tracrRNA plasmid 2.5ug and 1.5ul ODN which are constructed to target different gene sites into HEK293T cells with good states, and collecting all cells after 72h to extract DNA;

(e) performing ODN-PCR (ODN-polymerase chain reaction) by designing a pair of primers near an RNF2 targeted gene locus and on an ODN sequence, and preliminarily identifying whether a targeted cutting event occurs or not by observing whether a band exists or not through agarose gel electrophoresis;

(f) sanger sequencing verifies that ODN is successfully inserted into a target site, and confirms that the FrCas9 has the capacity of editing target DNA in eukaryotic cells;

the ODN-PCR result is shown in FIG. 6, panel A, and NGTA, NATA, NCTA, NTTA all have target bands with correct band size; the Sanger sequencing result is shown in FIG. 8, the ODN insertion position occurs in 3-4bp bases upstream of PAM, and the result shows that the range of target gene sequence cleavage of the FrCas 9and the previous SpCas9 is consistent.

Example 6

In this example, the optimal length of the sgRNA recognition part in the eukaryotic cell in the CRISPR/Cas9 gene editing system of Faecalibaculumrodentium type 2 described in the present invention is verified through ODN experiments, and the result is shown in fig. 7.

(1) Materials: all amino acid sequences, sgRNA sequences and identified PAMs of the Faecalibaculumrodentium editing genes obtained in examples 1 to 6;

(2) the verification method comprises the following steps: in this embodiment, ODN experiments are performed to verify that the optimal length of the sgRNA recognition part in the eukaryotic cell in the CRISPR/Cas9 gene editing system type 2 of Faecalibaculumrodentium provided by the invention,

the specific operation is as follows:

(a) synthesizing an adult-optimized FrCas9 protein sequence, cloning the protein sequence into a PX330 eukaryotic vector, and constructing a PX330-FrCas9 plasmid;

(b) selecting 30bp at the upstream of GGTA near a SpCas9 target site which is found to have better cutting effect in the previous research as a target sequence, constructing sgRNA with the recognition length of 19-23bp by a Gibson method, adding a human eukaryotic strong promoter U6 at the upstream of the sgRNA, and constructing PX330-FrCas9-sgRNA plasmid;

(c) electrotransfering the PX330-FrCas9-sgRNA plasmid 2.5ug and 1.5ul ODN which are constructed and target different gene sites into HEK293T cells with good state, and collecting all cells after 72h to extract DNA;

(d) a pair of primers designed near the target gene locus and on an ODN sequence is used for ODN-PCR, agarose gel electrophoresis is used for observing whether a band is present or not to preliminarily identify whether a target cutting event is generated or not, and the band strength of the sgRNA with the recognition length of 19-23bp is compared.

(e) Building an amplicon high-throughput library, quantifying the Indel rate of a target region, and comparing the cutting effect of the sgRNA with the recognition length of 19-23 bp;

as shown in fig. 7, the optimal sgRNA recognition length of FrCas9 is 21bp, 22bp, or 23 bp.

Example 7

In this example, the optimal length of the sgRNA framework in the eukaryotic cell in the CRISPR/Cas9 gene editing system of Faecalibaculumrodentium type 2 described in the present invention is verified through ODN experiments, and the result is shown in fig. 8.

(1) Materials: all amino acid sequences, sgRNA sequences and identified PAMs of the Faecalibaculumrodentium editing genes obtained in examples 1 to 6;

(2) the verification method comprises the following steps: in this embodiment, ODN experiments are performed to verify that the optimal length of sgRNA framework in eukaryotic cells in the CRISPR/Cas9 gene editing system type 2 of Faecalibaculumrodentium provided by the invention,

the specific operation is as follows:

(a) synthesizing an adult-optimized FrCas9 protein sequence, cloning the protein sequence into a PX330 eukaryotic vector, and constructing a PX330-FrCas9 plasmid;

(b) selecting 30bp at the upstream of GGTA near a SpCas9 target site which is found to have better cutting effect in the previous research as a target sequence, constructing sgRNA with the framework length of 71nt-95nt by a Gibson method, adding a human eukaryotic strong promoter U6 at the upstream of the sgRNA, and constructing PX330-FrCas9-sgRNA plasmid;

(c) electrotransfering the PX330-FrCas9-sgRNA plasmid 2.5ug and 1.5ul ODN which are constructed and target different gene sites into HEK293T cells with good state, and collecting all cells after 72h to extract DNA;

(d) a pair of primers designed near the target gene locus and on an ODN sequence is used for ODN-PCR, agarose gel electrophoresis is used for observing whether a band is present or not to preliminarily identify whether a target cutting event is generated or not, and the band strength of the sgRNA with the framework length of 71nt to 95nt is compared.

(e) Building an amplicon high-throughput library, quantifying the Indel rate of a target region, and comparing the cutting effect of sgRNA with the framework length of 71nt-95 nt;

as shown in fig. 8 and 3, the optimal sgRNA backbone for FrCas9 is 83nt-91 nt.

Example 8

In this embodiment, an ODN experiment verifies that the cleavage effect of the Faecalibaculumrodentium type 2 CRISPR/Cas9 gene editing system in eukaryotic cells is higher than SpCas9, and the off-target rate is lower than SpCas 9.

(1) Materials: all amino acid sequences, 22bp sgRNA sequence, 5 '-GGTA-3' PAM sequence, SpCas9 amino acid sequence, SpCas9 sgRNA sequence and SpCas 95 '-NGG-3' PAM sequence of the Faecalibachum editorium genes obtained in the embodiments 1-6;

(2) the verification method comprises the following steps: in this embodiment, a GUIDE-seq experiment verifies that the cutting effect of the Faecalibacculumrodentium type 2 CRISPR/Cas9 gene editing system in eukaryotic cell nucleus cells is higher than that of SpCas9, and the off-target rate is lower than that of SpCas 9.

The specific operation is as follows:

(a) synthesizing an adult-optimized FrCas9 protein sequence, cloning the protein sequence into a PX330 eukaryotic vector, and constructing a PX330-FrCas9 plasmid;

(b) selecting 30bp of GGTA upstream near a SpCas9 target site which is found to have better cutting effect in the previous research as a target sequence, constructing sgRNA with 22bp recognition length by a Gibson method, adding a human eukaryotic strong promoter U6 at the sgRNA upstream, and constructing PX330-FrCas9-sgRNA plasmid; simultaneously constructing PX330-SpCas9-sgRNA plasmid of 20bp SpCas9 sgRNA;

(c) the PX330-FrCas9-sgRNA and PX330-SpCas9-sgRNA plasmid 2.5ug which are constructed to target different gene sites and 1.5ul ODN are electrotransferred into HEK293T cells with good states, and all the cells are collected after 72h to extract DNA;

(d) a pair of primers designed near the target gene locus and on an ODN sequence is used for ODN-PCR, agarose gel electrophoresis is used for observing whether a band is present or not to preliminarily identify whether a target cutting event is generated or not, and the DNA with the band is subjected to GUIDE-seq library construction.

(e) Comparing the target-on cleavage effect and off-target condition of the same sites SpCas 9and FrCas9 through bioinformatics analysis;

as shown in fig. 9, FrCas9 was at DYRK1A-T2 at target Reads position 3257, higher than SpCas9 at target Reads 2456, while FrCas9 did not detect off-target and SpCas9 had 3-site off-target; FrCas9 has a GRIB2B-T9 site at target Reads number 34970, higher than SpCas9 at target Reads 20434, while FrCas9 has no detectable off-target and SpCas9 has 3 sites of off-target. The above data indicate that FrCas9 is a Cas9 protein with better cleavage efficiency and specificity than SpCas 9.

Sequence listing

<110> Zhuhaishutong medical science and technology Limited

<120> type 2 CRISPR/Cas9 gene editing system and application thereof

<160> 14

<170> SIPOSequenceListing 1.0

<210> 2

<211> 1372

<212> PRT

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 2

Met Cys Thr Lys Glu Ser Glu Lys Leu Asn Lys Asn Ala Asp Tyr Tyr

1 5 10 15

Ile Gly Leu Asp Met Gly Thr Ser Ser Ala Gly Trp Ala Val Ser Asp

20 25 30

Ser Glu Tyr Asn Leu Ile Arg Arg Lys Gly Lys Asp Leu Trp Gly Val

35 40 45

Arg Gln Phe Glu Glu Ala Lys Thr Ala Ala Glu Arg Arg Gly Phe Arg

50 55 60

Val Ala Arg Arg Arg Lys Gln Arg Gln Gln Val Arg Asn Arg Leu Leu

65 70 75 80

Ser Glu Glu Phe Gln Asn Glu Ile Thr Lys Ile Asp Ser Gly Phe Leu

85 90 95

Lys Arg Met Glu Asp Ser Arg Phe Val Ile Ser Asp Lys Arg Val Pro

100 105 110

Glu Lys Tyr Thr Leu Phe Asn Asp Ser Gly Tyr Thr Asp Val Glu Tyr

115 120 125

Tyr Asn Gln Tyr Pro Thr Ile Tyr His Leu Arg Lys Ala Leu Ile Glu

130 135 140

Ser Asn Glu Arg Phe Asp Ile Arg Leu Val Phe Leu Gly Ile His Ser

145 150 155 160

Leu Phe Gln His Pro Gly His Phe Leu Asp Lys Gly Asp Val Asp Thr

165 170 175

Asp Asn Thr Gly Pro Glu Glu Leu Ile Gln Phe Leu Glu Asp Cys Met

180 185 190

Asn Glu Ile Gln Ile Ser Ile Pro Leu Val Ser Asn Gln Lys Val Leu

195 200 205

Thr Asp Ile Leu Thr Asp Ser Arg Ile Thr Arg Arg Asp Lys Glu Gln

210 215 220

Gln Ile Leu Glu Ile Leu Gln Pro Asn Lys Glu Ser Lys Lys Ala Val

225 230 235 240

Ser Gln Phe Val Lys Val Leu Thr Gly Gln Lys Ala Lys Leu Gly Asp

245 250 255

Leu Ile Met Met Glu Asp Lys Asp Thr Glu Glu Tyr Lys Tyr Ser Phe

260 265 270

Ser Phe Arg Glu Lys Thr Leu Glu Glu Ile Leu Pro Asp Ile Glu Gly

275 280 285

Val Ile Asp Gly Leu Ala Leu Glu Tyr Ile Glu Ser Ile Tyr Ser Leu

290 295 300

Tyr Ser Trp Ser Leu Leu Asn Ser Tyr Met Lys Asp Thr Leu Thr Gly

305 310 315 320

His Tyr Tyr Ser Tyr Leu Ala Glu Ala Arg Val Ala Ala Tyr Asp Lys

325 330 335

His His Ser Asp Leu Val Lys Leu Lys Thr Leu Phe Arg Glu Tyr Ile

340 345 350

Pro Glu Glu Tyr Asp Asn Phe Phe Arg Lys Met Glu Lys Ala Asn Tyr

355 360 365

Ser His Tyr Ile Gly Ser Thr Glu Tyr Asp Gly Glu Lys Arg Cys Arg

370 375 380

Thr Ala Lys Ala Lys Gln Glu Asp Phe Tyr Lys Ser Ile Asn Lys Met

385 390 395 400

Leu Glu Lys Ile Pro Glu Cys Ser Glu Lys Thr Glu Ile Gln Lys Glu

405 410 415

Ile Ile Glu Gly Thr Phe Leu Leu Lys Gln Thr Gly Pro Gln Asn Gly

420 425 430

Phe Val Pro Asn Gln Leu Gln Leu Lys Glu Leu Arg Lys Ile Leu Gln

435 440 445

Asn Ala Ser Lys His Tyr Pro Phe Leu Thr Glu Lys Asp Glu Arg Asp

450 455 460

Met Thr Ala Ile Asp Arg Ile Glu Ala Leu Phe Ser Phe Arg Ile Pro

465 470 475 480

Tyr Tyr Ile Gly Pro Leu Lys Asn Thr Asp Asn Gln Gly His Gly Trp

485 490 495

Ala Val Arg Arg Asp Gly His Glu Gln Ile Pro Val Arg Pro Trp Asn

500 505 510

Phe Glu Glu Ile Ile Asp Glu Ser Ala Ser Ala Asp Leu Phe Ile Lys

515 520 525

Asn Leu Val Asn Ser Cys Thr Tyr Leu Arg Thr Glu Lys Val Leu Pro

530 535 540

Lys Ser Ser Leu Leu Tyr Gln Glu Phe Glu Val Leu Asn Glu Leu Asn

545 550 555 560

Asn Leu Arg Ile Asn Gly Met Tyr Pro Asp Glu Ile Gln Pro Gly Leu

565 570 575

Lys Arg Met Ile Phe Glu Gln Cys Phe Tyr Ser Gly Lys Lys Val Thr

580 585 590

Gly Lys Lys Leu Gln Leu Phe Leu Arg Ser Val Leu Thr Asn Ser Ser

595 600 605

Thr Glu Glu Phe Val Leu Thr Gly Ile Asp Lys Asp Phe Lys Ser Ser

610 615 620

Leu Ser Ser Tyr Lys Lys Phe Cys Glu Leu Phe Gly Val Lys Thr Leu

625 630 635 640

Asn Asp Thr Gln Lys Val Met Ala Glu Gln Ile Ile Glu Trp Ser Thr

645 650 655

Val Tyr Gly Asp Ser Arg Lys Phe Leu Lys Arg Lys Leu Glu Asp Asn

660 665 670

Tyr Pro Glu Leu Thr Asp Gln Gln Ile Arg Arg Ile Ala Gly Phe Lys

675 680 685

Phe Ser Glu Trp Gly Asn Leu Ser Arg Ala Phe Leu Glu Met Glu Gly

690 695 700

Tyr Lys Asp Glu Ala Gly Asn Pro Val Thr Ile Ile Arg Ala Leu Arg

705 710 715 720

Asp Thr Gln Lys Asn Leu Met Gln Leu Leu Ser Asn Asp Ser Ala Phe

725 730 735

Ala Lys Lys Leu Gln Glu Leu Asn Asp Tyr Val Thr Arg Asp Ile Trp

740 745 750

Ser Ile Glu Pro Asp Asp Leu Asp Gly Met Tyr Leu Ser Ala Pro Val

755 760 765

Arg Arg Met Ile Trp Gln Thr Phe Leu Ile Leu Arg Glu Val Val Asp

770 775 780

Thr Ile Gly Tyr Ser Pro Lys Lys Ile Phe Met Glu Met Ala Arg Gly

785 790 795 800

Glu Gln Glu Lys Lys Arg Thr Ala Ser Arg Lys Lys Gln Leu Ile Asp

805 810 815

Leu Tyr Lys Glu Ala Gly Met Lys Asn Asp Glu Leu Phe Gly Asp Leu

820 825 830

Glu Ser Leu Glu Glu Ala Gln Leu Arg Ser Lys Lys Leu Tyr Leu Tyr

835 840 845

Phe Arg Gln Met Gly Arg Asp Ile Tyr Ser Gly Lys Leu Ile Asp Phe

850 855 860

Met Asp Val Leu His Gly Asn Arg Tyr Asp Ile Asp His Ile His Pro

865 870 875 880

Gln Ser Lys Lys Lys Asp Asp Ser Leu Glu Asn Asn Leu Val Leu Thr

885 890 895

Ser Lys Asp Phe Asn Asn His Ile Lys Gln Asp Val Tyr Pro Ile Pro

900 905 910

Glu Gln Ile Gln Ser Arg Gln Lys Gly Phe Trp Ala Met Leu Leu Lys

915 920 925

Gln Gly Phe Met Ser Gln Glu Lys Tyr Asn Arg Leu Met Arg Thr Thr

930 935 940

Pro Phe Thr Asp Glu Glu Leu Ala Glu Phe Val Asn Arg Gln Leu Val

945 950 955 960

Glu Thr Arg Gln Gly Thr Lys Ala Ile Ile Ser Leu Ile Asn Gln Cys

965 970 975

Phe Pro Asp Ser Glu Val Val Tyr Val Lys Ala Gly Asn Thr Ser Asp

980 985 990

Phe Arg Gln Arg Phe Asp Ile Pro Lys Ser Arg Asp Leu Asn Asn Tyr

995 1000 1005

His His Ala Val Asp Ala Tyr Leu Asn Ile Val Val Gly Asn Val Tyr

1010 1015 1020

Asp Thr Lys Phe Thr Lys Asn Pro Ile Asn Phe Ile Lys Lys Met Arg

1025 1030 1035 1040

Lys Ser Gly Asn Leu His Ser Tyr Ser Leu Arg Arg Met Tyr Asp Phe

1045 1050 1055

Asn Val Gln Arg Gly Asp Gln Thr Ala Trp Val Ala Glu Asn Asp Thr

1060 1065 1070

Thr Leu Lys Thr Val Lys Lys Thr Ala Phe Lys Thr Ser Pro Met Val

1075 1080 1085

Thr Lys Arg Thr Tyr Glu Arg Lys Gly Gly Leu Ala Asp Ser Val Leu

1090 1095 1100

Ile Ala Ala Lys Lys Ala Lys Pro Gly Val His Leu Pro Val Lys Thr

1105 1110 1115 1120

Ser Asp Ser Arg Phe Ala Asn Gln Val Ser Thr Tyr Gly Gly Tyr Asp

1125 1130 1135

Asn Val Lys Gly Ser His Phe Phe Leu Val Glu His Gln Gln Lys Lys

1140 1145 1150

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

1155 1160 1165

Leu Lys Thr Lys Glu Glu Leu Glu His Tyr Cys Ala Gln Val Leu Gly

1170 1175 1180

Met Val Gln Pro Asp Val Arg Leu Thr Arg Ile Pro Met Tyr Ser Leu

1185 1190 1195 1200

Leu Leu Ile Asp Gly Tyr Tyr Tyr Tyr Leu Thr Gly Arg Thr Gly Gly

1205 1210 1215

Asn Leu Ser Leu Ser Asn Ala Val Glu Leu Cys Leu Pro Ala Lys Glu

1220 1225 1230

Gln Ala His Ile Arg Met Ile Ser Lys Ile Ala Gly Gly Arg Ser Thr

1235 1240 1245

Asp Ala Leu Ser Ala Glu Ala Lys Asp Asp Phe Arg Lys Lys Asn Leu

1250 1255 1260

Arg Leu Tyr Asp Glu Leu Ala Glu Lys His Arg Ser Thr Ile Phe Ser

1265 1270 1275 1280

Lys Arg Lys Asn Pro Ile Gly Pro Lys Leu Leu Lys Tyr Arg Glu Ala

1285 1290 1295

Phe Val Lys Gln Thr Ile Glu Asn Gln Cys Lys Val Ile Leu Gln Ile

1300 1305 1310

Leu Lys Leu Thr Ser Thr Asn Cys Lys Thr Ser Ala Asp Leu Lys Leu

1315 1320 1325

Ile Gly Gly Ser Gly Gln Glu Gly Val Met Ser Ile Ser Lys Leu Leu

1330 1335 1340

Arg Ala Glu Lys Tyr Ala Glu Phe Tyr Leu Ile Cys Gln Ser Pro Ser

1345 1350 1355 1360

Gly Ile Tyr Glu Thr Arg Lys Asn Leu Leu Thr Ile

1365 1370

<210> 2

<211> 294

<212> PRT

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 2

Met Thr Trp Arg Thr Ile Thr Ile Ser Ser His Ser Lys Leu Asp Tyr

1 5 10 15

Gln Met Gly Tyr Leu Val Val Arg Gly Glu Ser Ile Lys Arg Ile His

20 25 30

Leu Ser Glu Ile Ser Val Leu Ile Ile Glu Asn Thr Ala Val Ser Leu

35 40 45

Thr Ala Tyr Leu Val Ser Glu Leu Val Lys Asn Lys Ile Lys Leu Leu

50 55 60

Phe Cys Asp Glu Lys Arg Ser Pro Leu Ala Glu Val Ser Glu Leu Tyr

65 70 75 80

Gly Gly His Asp Ser Ser Asp Met Val Arg Lys Gln Ile Glu Ile Pro

85 90 95

Gln Glu Arg Lys Asp Ile Ala Trp Gln Ser Ile Ile Met Ser Lys Ile

100 105 110

Ser Asn Gln Phe Ala Val Leu His Asn Phe Asp Cys Pro Asn Gln Glu

115 120 125

Leu Leu Leu Gln Tyr Ile Asn Glu Val Leu Pro Gly Asp Val Thr Asn

130 135 140

Arg Glu Gly His Ala Ala Lys Val Tyr Phe Asn Ser Leu Phe Gly Lys

145 150 155 160

Ser Phe Tyr Arg Ala Ser Glu Cys Ala Leu Asn Ala Ala Leu Asn Tyr

165 170 175

Gly Tyr Ser Val Leu Leu Ser Ala Val Ser Arg Glu Ile Ala Gly Tyr

180 185 190

Gly Phe Leu Thr Gln Leu Gly Ile Phe His Asp Asn Cys Asp Asn Lys

195 200 205

Tyr Asn Leu Ser Cys Asp Leu Met Glu Pro Phe Arg Pro Val Val Asp

210 215 220

Tyr Leu Val Lys Ser Asn Ile Val Glu Val Phe Glu Lys Glu Gln Lys

225 230 235 240

Gln Lys Ile Leu Gln Leu Leu Gln Phe Lys Ile Gln Ile Asn Asp Arg

245 250 255

Gln Glu Thr Val Gln Asn Ala Ile Ser Ile Phe Val His Ser Val Leu

260 265 270

Asp Tyr Leu Leu Asp Pro Ser Val Tyr Ile Lys Val Pro Arg Ile Asp

275 280 285

Phe Thr Lys Asn Val Val

290

<210> 3

<211> 68

<212> PRT

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 3

Met Met Gln Glu Ser Val Tyr Cys Lys Leu Thr Thr Asn Gln Ser Ser

1 5 10 15

Ala Glu Thr Val Leu Lys Met Val Arg Ala Asn Lys Pro Pro Glu Gly

20 25 30

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

35 40 45

Phe Ile Leu Gly Gln Pro Asn Ser Asp Val Val Ala Thr Asp Glu Ser

50 55 60

Val Leu Asp Leu

65

<210> 4

<211> 223

<212> PRT

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 4

Met Arg Leu Leu Ile Asp Arg Leu Leu Leu Ser Ala Glu Leu Asn Ile

1 5 10 15

Asp Lys Ala Thr Thr Ile Ile Ile Glu Asn Pro Lys Ala Phe Arg Met

20 25 30

Val Ile Lys Asp Leu Ile Glu Gln Glu Asn Gly Gln Gly Gly Leu Leu

35 40 45

Arg Ile Val Glu Gly Asp Lys Glu Leu Cys Leu Ser Lys Ser Ala Ile

50 55 60

Leu Val Leu Asn Pro Tyr Leu Ala Asp Leu Asn Cys Arg Lys Phe Leu

65 70 75 80

Gln Leu Ala Tyr Ser Glu Leu Gln Ala Met Thr Gly Glu Phe Leu Glu

85 90 95

Asp Gln Ala Val Val Leu Ser Ala Met Thr Gly Tyr Leu Ser Lys Ile

100 105 110

Cys Asp Gln Ser Arg Phe Asp Phe Leu Glu Phe Ser Ala Ile Pro Asp

115 120 125

Trp Ala Ser Val Phe Lys Ala Trp Gly Leu Arg Phe Glu Gln Ala Ile

130 135 140

Pro Gly Leu Leu Pro Ser Leu Ile Gln Tyr Leu Gln Leu Ala Ala Thr

145 150 155 160

Phe Pro Gln Phe Lys Leu Ile Ile Phe Ile Asn Leu Lys Gln Tyr Leu

165 170 175

Leu Pro Glu Glu Gln Phe Glu Leu Phe Lys Met Ala Glu Tyr Leu Gln

180 185 190

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

195 200 205

Glu Asp Leu Ile Ile Ile Asp Lys Asp Leu Cys Glu Ile Gln Ser

210 215 220

<210> 5

<211> 20

<212> RNA

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 5

guuugagugu cuuguuaauu 20

<210> 6

<211> 36

<212> DNA

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 6

gtttgagtgt cttgttaatt cggaagtatt tcaaac 36

<210> 7

<211> 216

<212> DNA

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 7

gtttgagtgt cttgttaatt cgggagtagc tctctcgttt gagtgtcttg ttaattcgga 60

agtaagctca acttttgagt gtcttgttaa ttcggaagta tctcaaacgt ttgagtgtct 120

tgttaattca gaagtatttc aaacgtttga gtgtcttgtt aattcggaag tattccaaac 180

gtttgagtgt cttgttaatt cggaagtatt tcaaac 216

<210> 8

<211> 71

<212> RNA

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 8

aauuaacaag augaguucaa aucaggcucc uagagagauc cgaacuuacc uucauggcgg 60

gcauugugcc c 71

<210> 9

<211> 95

<212> RNA

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 9

guuugagugu cuuguuaauu gaaaaauuaa caagaugagu ucaaaucagg cuccuagaga 60

gauccgaacu uaccuucaug gcgggcauug ugccc 95

<210> 10

<211> 91

<212> RNA

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 10

guuugagugu cuuguuaaga aauuaacaag augaguucaa aucaggcucc uagagagauc 60

cgaacuuacc uucauggcgg gcauugugcc c 91

<210> 11

<211> 89

<212> RNA

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 11

guuugagugu cuuguuagaa auaacaagau gaguucaaau caggcuccua gagagauccg 60

aacuuaccuu cauggcgggc auugugccc 89

<210> 12

<211> 87

<212> RNA

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 12

guuugagugu cuuguugaaa aacaagauga guucaaauca ggcuccuaga gagauccgaa 60

cuuaccuuca uggcgggcau ugugccc 87

<210> 13

<211> 85

<212> RNA

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 13

guuugagugu cuugugaaaa caagaugagu ucaaaucagg cuccuagaga gauccgaacu 60

uaccuucaug gcgggcauug ugccc 85

<210> 14

<211> 83

<212> RNA

<213> Faecalibacilum rodentium bacterium (Faecalibacilum rodentium)

<400> 14

guuugagugu cuuggaaaca agaugaguuc aaaucaggcu ccuagagaga uccgaacuua 60

ccuucauggc gggcauugug ccc 83

Claims (10)

1. A type 2 CRISPR/Cas9 gene editing system, comprising a Cas9 protein, an accessory protein, a crRNA: CRISPR RNA and tracrRNA: transactivation CRISPR RNA in the form of a complex of Cas9 protein and guide RNA formed by the binding of crRNA, tracrRNA-FrCas 9 protein complex;

the Cas9 protein is a DNA endonuclease, and the Cas9 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.

2. The type 2 CRISPR/Cas9 gene editing system of claim 1, wherein the Cas9 protein cleaves double-stranded DNA complementary to sgRNA upstream of the PAM sequence through a different nuclease domain; the different nuclease domains are HNH-like nuclease domains or RuvC-like nuclease domains.

3. The type 2 CRISPR/Cas9 gene editing system of claim 1, wherein the auxiliary proteins comprise a Cas1 auxiliary protein, a Cas2 auxiliary protein and a Csn2 auxiliary protein;

the Cas1 helper protein has an amino acid sequence shown as SEQ ID NO. 2, or an amino acid sequence at least 80% similar to the amino acid sequence shown as SEQ ID NO. 2;

the Cas2 helper protein has an amino acid sequence shown as SEQ ID NO. 3, or an amino acid sequence at least 80% similar to the amino acid sequence shown as SEQ ID NO. 3;

the Csn2 accessory protein has an amino acid sequence shown in SEQ ID NO. 4 or an amino acid sequence which is at least 80% similar to the amino acid sequence shown in SEQ ID NO. 4.

4. The type 2 CRISPR/Cas9 gene editing system of claim 1, wherein the CRISPR RNA is generated by CRISPRArray transcription, and the CRISPR RNA has the RNA sequence shown in SEQ ID No. 5, or an RNA sequence at least 80% similar to the nucleic acid sequence of SEQ ID No. 5; the CRISPR Array comprises a direct repetitive sequence and a spacer sequence, wherein the direct repetitive sequence has a nucleic acid sequence shown in SEQ ID NO. 6 or a nucleic acid sequence which is at least 80 percent similar to the nucleic acid sequence shown in SEQ ID NO. 6; the spacer sequence has the nucleic acid sequence shown in SEQ ID NO. 7 or a nucleic acid sequence at least 80% similar to the nucleic acid sequence of SEQ ID NO. 7.

5. The type 2 CRISPR/Cas9 gene editing system of claim 1, wherein the transactivation CRISPR RNA comprises a sequence complementary to the CRISPR RNA direct repeat and the transactivation CRISPR RNA has the nucleic acid sequence shown in SEQ ID No. 8 or a nucleic acid sequence at least 70% similar to the nucleic acid sequence shown in SEQ ID No. 8.

6. The type 2 CRISPR/Cas9 gene editing system according to claim 1, wherein the crRNA and tracrRNA are combined to form a guide RNA whose backbone consists of the sequence of the 7-24nt part of the crRNA direct repeat and the tracrRNA sequence; the two parts can be fused by a "GAAA", "TGAA" and "AAAC" linker to form a sgRNA framework;

preferably, the sgRNA backbone formed by fusion of the 20nt direct repeat sequence and the tracrRNA full-length sequence by "GAAA" is shown in SEQ ID No. 9, and on this basis, the results of highly efficient variants of the sgRNA backbone preferably include the following 5:

the sgRNA backbone formed by fusion of the first 18-14nt of the direct repeat sequence of the crRNA with the last 69-65nt of the tracrRNA by the "GAAA" linker sequence:

sgRNA backbone length 91 nt: 18nt direct repeat +69nttracrRNA, as shown in SEQ ID NO: 10;

sgRNA backbone length 89 nt: a 17nt direct repeat +68nttracrRNA as shown in SEQ ID NO: 11;

length of sgRNA backbone 87 nt: 16nt direct repeat +67nttracrRNA, as shown in SEQ ID NO: 12;

length of sgRNA backbone 85 nt: 15nt direct repeat +66nttracrRNA, shown in SEQ ID NO: 13;

sgRNA backbone length 83 nt: 14nt direct repeat +65nttracrRNA, shown in SEQ ID NO: 14;

the nucleic acid sequences shown above include, but are not limited to, nucleic acid sequences that are at least 70% similar to SEQ ID NOS 9-14.

7. The type 2 CRISPR/Cas9 gene editing system according to claim 1, wherein the type 2 CRISPR/Cas9 gene editing system derived from Faecalibacculumrodentium binds or cleaves specific DNA in biological process, the binding or cleavage of specific DNA is realized by complementary pairing recognition of guide RNA and specific DNA target, and the length of the guide RNA pairing binding part is in the range of 14-30bp nucleotide length;

the specific DNA is DNA of prokaryotes or eukaryotes.

8. The type 2 CRISPR/Cas9 gene editing system according to claim 7, wherein the length of the pairing-binding moiety recognized by the complementary pairing of the guide RNA and the specific DNA target is 21bp, 22bp or 23bp, and wherein the Cas9 protein complex is highly sensitive to a 14bp base mismatch near the adjacent module of the original spacer sequence and is a seed region.

9. The type 2 CRISPR/Cas9 gene editing system of claim 7, wherein the protospacer proximity module sequence required for binding or cleaving DNA function is 5 '-NNTA-3' downstream of the guide RNA/sgRNA recognition sequence, wherein N is A, T, C or G.

10. Use of a type 2 CRISPR/Cas9 gene editing system as claimed in any of claims 1-9 for editing or binding prokaryotic or eukaryotic DNA.

Images