CN113234701A - Cpf1 protein and gene editing system - Google Patents

Abstract

The invention relates to a Cpf1 protein and a gene editing system, which comprise a Cpf1 protein or one or more polynucleotides encoding the Cpf1 protein, and CRISPR RNA or one or more polynucleotides encoding the CRISPR RNA protein; wherein, the amino acid sequence of the Cpf1 protein is shown as SEQ ID NO.1 or a sequence with at least 80% homology with SEQ ID NO. 1. According to the invention, a new type 2 CRSIPR/Cas gene editing system is excavated through the bioinformatics analysis of the 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. The invention provides a novel V-type CRISPR/Cas12a gene editing system which has novel physicochemical properties and can identify various different PAM sequences (TTNA).

Description

Cpf1 protein and gene editing system

Technical Field

The invention belongs to the technical field of gene editing, and particularly relates to a Cpf1 protein and a gene editing system.

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.

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) system is a natural immune system from archaea and bacteria, is a third-generation gene editing tool, is different from the conventional gene editing tool (protein-DNA recognition), utilizes the complementary pairing principle of nucleic acid base to recognize a target DNA sequence, guides a Cas effector protein to perform site-directed cutting, 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/Cas system derived from Streptococcus pyogenes (Streptococcus pyogene Cas9, SpCas9) is currently 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 recent years, type II CRISPRs comprising Cpf1, classified as type V, have also been increasingly used in the field of gene editing, and unlike Cas9 endonuclease, the cleavage of Cpf1 protein requires only a single RNA guide, which can simplify the design and use of gene editing tools. Meanwhile, Cpf1, a system that produces gene editing effects by recognizing T-rich PAM and leaving sticky ends in its targeting DNA sequence, the T/C preference of Cpf1 family proteins to recognize PAM expands the range of CRISPR targeted editing nucleic acids.

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

Disclosure of Invention

The object of the present invention is to provide a novel gene editing system to enrich the existing gene editing tool family.

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

according to a first aspect of the present invention there is provided a Cpf1 protein, the amino acid sequence of the Cpf1 protein being as shown in SEQ ID No.1 or having at least 80% homology thereto.

Preferably, the amino acid sequence of the Cpf1 protein has at least 85% homology with the amino acid sequence shown in SEQ ID No.1, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, 97%, 98%, 99% homology.

Preferably, the Cpf1 protein comprises one or more mutant amino acid residues at positions: positions 117, 150, 267, 281, 538, 593, 776.

The Cpf1 protein is a DNA endonuclease, and the Cpf1 protein cleaves double-stranded DNA complementary to CRISPR RNA (crRNA) downstream of the PAM sequence through different nuclease domains; the different nuclease domains are HNH-like nuclease domains or RuvC-like nuclease domains.

The Cpf1 protein is abbreviated as LtCpf1, comprises 1296 amino acids and is a multifunctional DNA multi-domain endonuclease. The DNA has RNA and DNA endonuclease activities simultaneously, participates in maturation of pre-crRNA, recognizes and effectively cuts double-stranded DNA which is complementary with the crRNA and is positioned downstream of PAM through a RuvC nuclease domain and an HNH-like nuclease domain, cuts 23 th nucleotide of a target DNA chain and 17 th nucleotide of a non-target DNA chain downstream of the PAM sequence, and generates a sticky end with 6 nucleotide protrusions.

In a second aspect, the present invention provides a polynucleotide encoding a Cpf1 protein as defined above.

Preferably, the polynucleotide is codon optimized for expression in the cell of interest.

In a third aspect, the present invention provides a vector comprising the polynucleotide as described above.

In a fourth aspect, the invention provides a vector system comprising one or more vectors comprising a polynucleotide as described above and comprising one or more polynucleotides encoding CRISPR RNA on the same or different vectors.

In a fifth aspect of the present invention there is provided a complex comprising the Cpf1 protein, and CRISPR RNA.

The sixth aspect of the present invention provides a V-type CRISPR/Cas12a gene editing system comprising said Cpf1 protein or one or more nucleotide sequences encoding said Cpf1 protein, and one or more polynucleotide sequences of CRISPR RNA encoding said CRISPR RNA.

Preferably, the V-type CRISPR/Cas12a gene editing system further comprises an accessory protein, which may be involved in the capture of a foreign gene, or one or more polynucleotides encoding the accessory protein.

Further preferably, the auxiliary protein comprises one or more of a Cas1 protein, a Cas2 protein and a Cas4 protein.

Further preferably, the Cas1 protein has the amino acid sequence shown in SEQ ID No.2, or an amino acid sequence at least 80% homologous to the amino acid sequence shown in SEQ ID No.2, preferably at least 85% homologous, further preferably at least 90% homologous, more preferably at least 95% homologous, still more preferably at least 96%, 97%, 98%, 99% homologous;

the Cas2 protein has an amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence at least 80% homologous with the amino acid sequence shown in SEQ ID NO.3, preferably at least 85% homologous, further preferably at least 90% homologous, more preferably at least 95% homologous, and still more preferably at least 96%, 97%, 98%, 99% homologous;

the Cas4 protein has an amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence at least 80% homologous with the amino acid sequence shown in SEQ ID NO.4, preferably at least 85% homologous, further preferably at least 90% homologous, more preferably at least 95% homologous, and still more preferably at least 96%, 97%, 98%, 99% homologous. The seventh aspect of the present invention also provides a design principle CRISPR RNA, including one or more of the following:

a) CRISPR RNA the sequence format is: 5 '-direct repeat binding to Cpf1 protein-spacer complementary to target sequence-3';

b) CRISPR RNA the length of the spacer sequence is 10-30 bases;

c) CRISPR RNA has a direct repeat sequence length of 12-37 bases;

d) CRISPR RNA should contain a stem-loop structure.

Specifically, the target sequence is an exogenous DNA fragment or a target sequence designed and artificially synthesized aiming at a target gene.

Preferably, the CRISPR RNA direct repeat sequence is as shown in SEQ ID NO.5 or has at least 80% homology thereto, more preferably the CRISPR RNA direct repeat sequence is as shown in any one of SEQ ID NO.6 to 12 or has at least 80% homology, preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95% homology, even more preferably at least 96%, 97%, 98%, 99% homology to any one of SEQ ID NO.6 to 12.

Preferably, CRISPR RNA is generated by CRISPR Array transcription, which results in precursor CRISPR RNA (pre-crRNA), precursor CRISPR RNA is processed and cleaved to form CRISPR RNA, wherein CRISPR RNA is used as a guide RNA to form a complex with Cpf1 protein, and the mature CRISPR RNA spacer sequence processed by transcription is complementary to the anchor gene of interest, which leads Cpf1 protein to cleave the gene in the genome of interest.

The CRISPR Array comprises a direct repetitive sequence matched with the Cpf1 protein and a spacer sequence, wherein the spacer sequence comprises a target sequence and an element related to the Cpf1 protein.

Further preferably, the sequence of the element related to the Cpf1 protein is as shown in one or more of SEQ ID NO 13 to NO 17 or a sequence having at least 80% homology with the sequence shown in SEQ ID NO 13 to NO 17, preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95% homology, even more preferably at least 96%, 97%, 98%, 99% homology.

CRISPR RNA (crRNA) according to the present invention, in a base complementary form, directs the Cpf1 protein to recognize the invading foreign genome. When bacteria are exposed to bacteriophage or virus and the like for invasion, short fragments of exogenous DNA are integrated between CRISPR array repeated spacer sequences in a host chromosome as new spacer sequences, thereby providing genetic record of infection, when the organism is invaded by exogenous genes again, CRISPR array is transcribed to generate precursor crRNA (pre-crRNA), precursor CRISPR RNA (pre-crRNA) is cut and processed to obtain mature CRISPR RNA (crRNA) with 5 'end being direct repeated sequence and 3' end being spacer sequence, the direct repeated sequence at 5 'end guides Cpf1 protein to be combined with a target sequence, the spacer sequence at 3' end is complementary to the sequence of the exogenous invasion genes, and mature CRISPR RNA (crRNA) is used as guide RNA (sgRNA) of Cpf1 protein to cut and target sequence.

The vector system, the complex, or the V-type CRISPR/Cas12a gene editing system in the invention binds or cuts the structure of DNA function in biological process; preferably, the structure of the DNA function includes, but is not limited to, a crRNA secondary structure, a Cpf1 effector protein domain, or a Cpf1-crRNA complex structure; preferably, the DNA is a prokaryotic or eukaryotic DNA.

The Cpf1 protein (LtCpf1) can recognize the break (DSB) of DNA double-stranded molecules formed by adjacent modules (PAM) of various protospacer sequences immediately upstream of a targeting sequence, and two important factors are required for recognizing the targeting sequence by the LtCpf1 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 LtCpf1 is shown to have a cutting effect in a prokaryotic system through a depletion experiment, and the positions 1 and 2 of the PAM sequence recognized by the newly discovered V-type CRISPR/Cas12a system are preliminarily verified to be TT. And interference experiments and in vitro cutting prove that the PAM upstream of the target sequence recognized by the LtCpf1 can be TTNA (N is A, T, C, G). By artificially designing the spacer sequence in the crRNA, this CRISPR-Cas system can target almost all DNA sequences of interest in the genome, creating site-specific cohesive-end 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. In addition to directional cleavage of double-stranded DNA, LtCpf1 activates the single-stranded DNA cleavage domain upon targeting the double-stranded DNA of interest, thereby generating a side-cut single-stranded DNA effect. The characteristic of the indiscriminate lateral cutting single-stranded DNA can be applied to the rapid nucleic acid detection of DNA or RNA viruses, and has remarkable clinical application value.

The vector system, the compound and the V-shaped CRISPR/Cas12a gene editing system are applied to editing or detecting genes of prokaryotes or eukaryotes; preferably, it is used for binding, targeted cleavage or non-targeted cleavage of DNA.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

(1) according to the invention, a new type 2 CRSIPR/Cas gene editing system is excavated through the bioinformatics analysis of the 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 novel V-shaped CRISPR/Cas12a gene editing system provided by the invention has novel physicochemical properties and can identify various different PAM sequences, and the PAM specific sequence identified by the gene editing system is TTNA (N represents A, T, C, G).

Drawings

Fig. 1 is a composition diagram of the homologous V-type CRISPR/Cas12a gene editing system described in the present invention.

FIG. 2 is a schematic diagram of sRNA-seq of the homologous V-type CRISPR/Cas12a gene editing system.

Fig. 3 is a schematic diagram of a preferred crRNA backbone of the homologous V-type CRISPR/Cas12a gene editing system described in the present invention.

Fig. 4 is a conserved PAM sequence of the homologous V-type CRISPR/Cas12a gene editing system of the present invention.

Fig. 5 shows the interference experiment result of the homologous V-type CRISPR/Cas12a gene editing system of the present invention.

Fig. 6 is a schematic diagram of the homologous type V CRISPR/Cas12a gene editing system of the present invention for targeted cleavage of target DNA at the in vitro level;

fig. 7 is a specific PCR experiment gel and Sanger sequencing result diagram of the homologous V-type CRISPR/Cas12a system of the present invention inserted into a double-stranded oligonucleotide chain (dsODN) after a eukaryotic cell line cuts a target.

Fig. 8 is a schematic diagram of the optimal length of the direct repeat sequence of the homologous V-type CRISPR/Cas12a gene editing system described in the present invention.

Fig. 9 is a schematic diagram of the optimal length of the recognition sequence of the homologous V-type CRISPR/Cas12a gene editing system described in the present invention.

FIG. 10 is a schematic diagram showing the result of the rapid nucleic acid detection of the COVID-19 gene by the homologous V-type CRISPR/Cas12a gene editing system of the present invention.

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.

Example 1

In the embodiment, the protein and related elements related to the CRISPR-Cas system are obtained by analyzing, predicting and screening intestinal metagenome; the V-type CRISPR/Cas12a gene editing system is composed as shown in figure 1.

As can be seen from fig. 1, the V-type CRISPR/Cas12a gene editing system described in the present invention comprises the following components: endonuclease Cpf1(LtCpf1), helper proteins Cas1, Cas2, Cas4, crRNA. The LtCpf1 comprises 1296 amino acids, the sequence of the LtCpf1 is shown as SEQ ID NO.1, and double-stranded DNA complementary to the sgRNA is effectively cut at the downstream of the PAM through different nucleotidase domains; the auxiliary proteins Cas1 (the sequence of which is shown in SEQ ID NO. 2), Cas2 (the sequence of which is shown in SEQ ID NO. 3), Cas4 (the sequence of which is shown in SEQ ID NO. 4) are involved in foreign gene capture and maturation of crRNA; the crRNA includes a direct repeat sequence (the sequence of which is shown in SEQ ID No.5) and a spacer sequence (complementary to the foreign gene fragment or the artificially designed target sequence), and the crRNA can be transcribed by the CRISPR Array, which includes the direct repeat sequence (corresponding to the direct repeat sequence of the crRNA) and the spacer sequence, and the spacer sequence of the CRISPR Array includes the foreign gene fragment or the artificially designed target sequence, the spacer sequence corresponding to the crRNA, and elements related to the LtCpf1 protein, as shown in SEQ ID nos. 13 to 17.

With the aid of the proteins Cas1, Cas2, Cas4, foreign gene fragments or artificially designed sequences (target sequences) are integrated as new spacer sequences between the direct repeats of the CRISPR Array, which transcribes to produce the precursor CRISPR RNA (pre-crRNA), the pre-crRNA sequence being from 5 'to 3': the spacer of the 5 '-direct repeat-spacer-direct repeat-3', pre-crRNA includes sequences complementary to the target sequence as well as sequences complementary to elements associated with the LtCpf1 protein. The pre-crRNA is then cut and processed to form a mature CRISPR RNA (crRNA) sequence with a direct repeat sequence at the 5 'end and a spacer sequence at the 3' end, the spacer sequence at the 3 'end is a sequence complementary to a foreign gene segment or an artificially designed sequence (target sequence), and the direct repeat sequence at the 5' end guides the LtCpf1 protein to be combined with the target sequence, so that the sequence can be used as a guide RNA (sgRNA) to guide the Lt1Cas13d protein to cut a target sequence.

Example 2

This example is to verify the crRNA structure of the V-type CRISPR/Cas12a gene editing system predicted in example 1 of the present invention.

(1) Materials: the CRISPR/Cas gene editing system-associated genes predicted in example 1 above.

(2) The verification method comprises the following steps: constructing a mature escherichia coli editing system by using the V-type CRISPR/Cas12a gene editing system, extracting total RNA of recombinant escherichia coli, purifying, adding a sequencing linker, and analyzing to obtain a wild mature CRISPR/Cas12a-crRNA structure;

the specific operation is as follows:

(a) inserting the V-type CRISPR/Cas12a gene editing system (comprising endonuclease LtCpf1, helper proteins Cas1, Cas2, Cas4 and CRISPR array) described in embodiment 1 of the invention into a pACYC184 vector, carrying out Escherichia coli codon optimization on Cpf1 protein, adding elements (SEQ ID NO. 13-NO. 17) related to LtCpf1 protein described in embodiment 1 and a Library sequence (SEQ ID NO.18) adopted in reference (1) into the CRISPR array as target sequences, and adding strong heterologous promoters J23119 on Ltprokaryotic 1 protein and CRISPR array to construct a plasmid for expressing pACYC184-LtCpf1, so as to obtain recombinant Escherichia coli;

(b) extracting total RNA of the recombinant escherichia coli, and performing DNA removal, rRNA removal and phosphorylation treatment on a sample;

(c) reserving sRNA with the length range of 15-120nt in an RNA sample, adding an illlumina platform sequencing joint on two sections of the sRNA, performing reverse transcription on an RNA library, performing PCR amplification, and performing high-throughput sequencing on the obtained high-yield sRNA-seq sequencing library;

(d) and (3) aligning the CRISPR/Cas12a sequence maps to obtain a wild mature CRISPR/Cas12a-crRNA structure.

FIG. 2 is a schematic diagram of sRNA-seq of the homologous V-type CRISPR/Cas12a gene editing system.

By comparing CRISPR/Cas12a sequence maps, as shown in fig. 2, under the action of LtCpf1 nuclease, pre-crRNA removes 15nt upstream of the repeat sequence and 7-9nt downstream of the spacer sequence, forming mature crRNA with a direct repeat sequence at the 5 'end and a spacer sequence at the 3' end, and the mature crRNA is fused with LtCpf1 to form a crRNA-Cpf1 complex.

Reference (1) is Evelt, K.M., Mali, P., Braff, J.L., Moosburn, M., Yaung, S.J.and Church, G.M, (2013) organic Cas9 proteins for RNA-regulated gene regulation and identification. Nat Methods,10,1116-1121.

Example 3

This example is to predict the secondary structure of crRNA of V-type CRISPR/Cas12a gene editing system described in example 1 of the present invention.

The combination process of RNA transcribed by a Spacer sequence (Spacer) and a direct repeat sequence (repeat sequence) is simulated, and the structure of crRNA after the combination of the Spacer sequence and the repeat sequence is predicted.

(1) Materials: predicted DR sequence and repeat sequence.

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

(3) The prediction method comprises the following steps: the secondary structure of CRISPR RNA (crRNA) was simulated in vitro at 37 ℃ by using on-line NUPACK application.

Fig. 3 is a schematic diagram of a preferred crRNA framework of a crRNA molecule identified by the homologous V-type CRISPR/Cas12a gene editing system of the present invention after screening and optimization. FIG. 3 shows that the repeat sequence of crRNA (shown in SEQ ID NO.5) forms a 5 base-paired stemloop secondary structure, which is the key domain that crRNA and LtCpf1 protein recognize each other and direct LtCpf1 protein to target double-stranded DNA cleavage. Also, fig. 3 shows that the variant structure of the repeat sequence (SEQ ID No.5) forming the stem-loop structure in the crRNA backbone preferably includes repeat sequences as shown in SEQ ID nos. 6 to 12.

Example 4

The purpose of this example is to discover a protospacer proximity module (PAM) recognized by the V-type CRISPR/Cas12a gene editing system of the present invention in a prokaryotic system.

(1) Materials: the pACYC184-LtCpf1 plasmid, the target-library plasmid, E.coli DH5a obtained in example 2.

(2) The verification method comprises the following steps: in this embodiment, a prokaryotic verification system is constructed for the V-type CRISPR/Cas12a gene editing system described in the present invention, the cleavage effect thereof is verified, and the identified PAM sequence is primarily discovered by a second-generation sequencing technology. .

The specific operation is as follows:

(a) adding 7 random bases (16384 insertion fragments in total) at the 3' end of the spacer sequence (SEQ ID NO.18) of the library, performing multiple cloning on the MCS of a pUC19 vector to select EcoRI and NcoI two enzyme cutting sites, cloning the library into the vector, and constructing a target-library plasmid;

(b) transfecting Escherichia coli DH5a with pACYC184-LtCpf1 plasmid or unloaded pACYC184 plasmid, making into competent cells, respectively transferring into 200ng target-library plasmid, recovering at 25 ℃ for 2h, uniformly coating on SOB culture medium containing ampicillin sodium (100ug/ml) and chloramphenicol (25ug/ml) dual resistance, incubating at 25 ℃ for 30h, and collecting the plasmid by alkaline lysis method;

(c) PCR amplification contains a spacer sequence and seven random bases, two ends of a PCR product are added with joints for second-generation sequencing, a PAM exhaustion threshold value (PPDV) relative to a no-load control group is calculated, and a PAM sequence of the V-type CRISPR/Cas12a gene editing system is generated by using Weblogo.

Fig. 4 is a schematic diagram of a conserved PAM sequence adjacent to a module of an original spacer sequence recognized by the V-type CRISPR/Cas12a 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 by Weblogo into LtCpf1 is TTNA.

Example 5

In this example, a plurality of possible protospacer proximity modules (PAMs) identified in the V-type CRISPR/Cas12a gene editing system described in example 1 of the present invention were further determined and verified by interference experiments, and their cleavage ability at a prokaryotic level was determined.

(1) Materials: pACYC184-LtCpf1 plasmid, target-library plasmid, PAM preliminarily recognized by LtCpf1 obtained in example 4, E.coli DH5 a.

(2) The verification method comprises the following steps: in this embodiment, an escherichia coli interference experiment is used to further determine a Protospacer Adjacent Module (PAM) recognized by the V-type CRISPR/Cas12a gene editing system in a prokaryotic system;

the specific operation is as follows:

(a) TTNA (N stands for A, T, C, G) is added at the 5 'end of the spacer sequence (SEQ ID NO.18), and the 5' end is cloned into pUC19 through EcoRI and NcoI enzyme cutting sites respectively to construct target-library plasmid with PAM as TTNA;

(b) the target-library plasmid containing TTNA as 5' terminal PAM was transferred into E.coli DH5a electrotransfer competence containing LtCpf 1-related locus, recovered at 25 ℃ for 2h, then diluted in gradient, incubated overnight at 25 ℃ on SOB medium containing ampicillin sodium (100ug/ml) and chloramphenicol (25ug/ml) dual-resistance by dot coating, and the number of monoclonal bacteria was observed.

FIG. 5 is a schematic diagram of an interference experiment of the V-type CRISPR/Cas12a gene editing system, PAMs of target-library plasmids used in the 1 st, 2 nd, 3 th and 4 th columns in FIG. 5 from left to right are TTAA, TTTA, TTCA and TTGA respectively, and the 5 th column is non-target plasmid transferred into LtCpf1 E.coli. The colony numbers of the monoclonals are observed by gradient dilution, and the colony numbers of the 1 st, 2 nd, 3 th and 4 th columns (TTNA-target) are obviously reduced compared with the colony number of the 5 th column (non-target), which indicates that the LtCpf1 can effectively identify TTNA as PAM and target and cut a DNA sequence in Escherichia coli.

Example 6

In this example, the in vitro cleavage experiment is performed to verify the in vitro cleavage capability and the potential gene editing capability in eukaryotes of the V-type CRISPR/Cas12a gene editing system described in example 1 of the present invention.

(1) Materials: PAM preliminarily identified by LtCpf1 obtained in example 4; the structure of LtCpf1 wild-type crRNA obtained by sRNA-seq; purifying LtCpf1 protein and HEK293T cell DNA in vitro;

(2) the verification method comprises the following steps: in this embodiment, an HEK293T cell DNA in vitro cleavage experiment is used to further determine whether the V-type CRISPR/Cas12a gene editing system of the present invention has the ability to cleave double-stranded DNA in a targeted manner at an in vitro level, and the identified protospacer proximity module (PAM);

the specific operation is as follows:

(a) selecting a sequence with TTNA near a target spot tested by a literature as an in vitro cutting target spot (SEQ ID NO. 19-NO. 22) to be added into a CRISPR array, and designing a specific primer to amplify to obtain an HEK293T cell DNA in vitro cutting template;

(b) CRISPR array transcribes 4 pieces of LtCpf1-crRNA aiming at 4 different PAMs (TTAA, TTTA, TTCA and TTGA) in vitro;

(c) according to LtCpf 1: crRNA: mixing samples with the molar weight of the template being 10:10:1, incubating for 15 minutes at 37 ℃, performing agarose gel electrophoresis, and observing whether the DNA template has a cutting effect.

Fig. 6 is a schematic diagram of in vitro cleavage of the V-type CRISPR/Cas12a gene editing system described in the present invention. Fig. 6 shows that LtCpf1 is effective in recognizing TTNA and performing targeted cleavage at in vitro levels, wherein significant double strand breaks are observed in the TTAA, TTTA, and TTCA groups, and relatively weak double strand breaks are observed in the TTGA group. The LtCpf1 can generate an effective targeted gene cleavage effect in vitro by combining a PAM sequence preliminarily discovered in a depletion experiment and a crRNA skeleton structure obtained by sRNA-seq.

Example 7

In this embodiment, the ability of the V-type CRISPR/Cas12a gene editing system of the present invention to cut a target DNA sequence in a eukaryotic cell is verified through an ODN experiment, and ODN-PCR results and Sanger sequencing results are shown in fig. 7.

(1) Materials: the CRISPR/Cas gene editing system of example 1 and PAM identified by LtCpf1 obtained in example 4, the existing V-type CRISPR/Cas12a gene editing system (LbCpf1 protein), LbCpf1 plasmid was purchased from addrene, the catalog number is PY 016;

(2) the verification method comprises the following steps: in this example, ODN experiments are performed to verify that the V-type CRISPR/Cas12a gene editing system of LtCpf1 provided by the invention has the capability of cutting a target DNA sequence in eukaryotic cells,

the specific operation is as follows:

(a) synthesizing an adult-optimized LtCpf1 protein sequence, cloning the LtCpf1 protein sequence into a PX330 eukaryotic vector, and constructing a PX330-LtCpf1 plasmid;

(b) selecting a human gene locus CDKN2A to design crRNA, wherein the design principle is as follows (refer to the sequencing result of sRNA-seq):

1) crRNA includes Spacer (Spacer) and Direct Repeat (DR) sequences in the format: 5 '-direct repeat-spacer-3' that binds to LtCpf1 protein;

2) the length of the spacer sequence of the crRNA is 10-30 base sequences; 3) the length of the direct repetitive sequence of the crRNA is 12-37 base sequences;

4) the direct repeat sequence of crRNA should contain a stem loop structure (stem loop);

inserting crRNA (the structural design of wild mature crRNA obtained by referring to sRNA-SEQ: the Direct Repeat length is 22bp, and the Spacer length is 23bp) into a target plasmid by a Gibson method according to a Spacer sequence designed by CDKN2A, such as SEQ ID NO.20, adding a human-derived eukaryotic strong promoter U6 at the upstream of the crRNA, and constructing a PX330-LtCpf1-crRNA plasmid;

(c) electrically transferring PX330-LbCpf1-crRNA plasmid 2.5ug and 1.5ul ODN of the constructed target gene locus CDKN2A into HEK293T cells with good state, and collecting all cells after 72h to extract DNA;

(d) a pair of primers near the target gene locus and on the ODN sequence are designed to carry out ODN-PCR, agarose gel electrophoresis is used for observing whether a band exists or not, and whether a target cutting event occurs or not is preliminarily identified.

As shown in FIG. 7, agarose gel electrophoresis showed that the positive control Lcpcf 1 and the experimental group Lcpcf 1 both had the ODN-PCR target band with the correct size and the band intensities were substantially the same. Sanger sequencing showed successful integration of the ODN fragment into the target gene CDKN2A targeting amino acid sequence.

Example 8

In this example, the optimal spacer sequence (DR) length of the target DNA sequence in eukaryotic cells in the V-type CRISPR/Cas12a gene editing system described in the present invention is verified through an amplicon experiment, and a histogram result of the quantitative editing efficiency of the amplicon is shown in fig. 8.

(1) Materials: CRISPR/Cas gene editing system of example 1 and PAM recognized by LtCpf1 obtained in example 4;

(2) the verification method comprises the following steps: this example compares the effect of different length spacer sequences (DR) in editing CDKN2A gene in eukaryotic cells by amplicon experiments

The specific operation is as follows:

(a) synthesizing an adult-optimized LtCpf1 protein sequence, cloning the LtCpf1 protein sequence into a PX330 eukaryotic vector, and constructing a PX330-LtCpf1 plasmid;

(b) the human gene locus CDKN2A is selected to design crRNA, and the design principle is as follows:

1) crRNA includes Spacer (Spacer) and Direct Repeat (DR) sequences in the format: 5 '-direct repeat-crRNA spacer-3' that binds to LtCpf1 protein;

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

3) the length of the direct repetitive sequence of the crRNA is 14, 16, 18, 20, 22, 24, 26 and 28 base sequences;

4) the direct repeat sequence of crRNA should contain a stem loop structure (stem loop);

4) respectively inserting 8 different crRNAs into a target plasmid by a Gibson method, adding a human eukaryotic strong promoter U6 at the upstream of the crRNAs, and constructing 8 PX330-LtCpf1-crRNA plasmids;

(c) respectively electrotransfering PX330-LbCpf1-crRNA plasmid 2.5ug and 1.5ul ODN of the constructed target human gene locus CDKN2A into HEK293T cells with good states, and collecting all cells after 72h to extract DNA;

(d) performing ODN-PCR by designing a pair of primers near the target gene locus and on the ODN sequence, observing a target band with correct size by agarose gel electrophoresis, and preliminarily identifying that a target cutting event occurs;

(e) designing a proper amplification primer aiming at a target gene sequence, carrying out amplicon high-throughput library construction on DNA, quantifying the Indel rate of a target region, and comparing the gene editing efficiency when DR is respectively 14, 16, 18, 20, 22, 24, 26 and 28 bases;

the amplicon quantitative gene editing efficiency histogram is shown in fig. 8. The results show that the crRNA can not effectively guide LtCpf1 to target genome editing when the DR is lower than 20nt, and can effectively guide LtCpf1 to target genome editing when the DR ranges from 22 nt to 28 nt.

Example 9

In this embodiment, the best recognition sequence (Spacer) length of the target DNA sequence in eukaryotic cells in the V-type CRISPR/Cas12a gene editing system described in the present invention is verified through an amplicon experiment, and a histogram result of amplicon quantitative editing efficiency is shown in fig. 9.

(1) Materials: the CRISPR/Cas gene editing system of the example and the resulting PAM recognized by LtCpf1 of example 4;

(2) the verification method comprises the following steps: in this embodiment, the optimal recognition sequence (Spacer) length of the target DNA sequence in eukaryotic cells in the V-type CRISPR/Cas12a gene editing system is verified by amplicon experiments,

the specific operation is as follows:

(a) synthesizing an adult-optimized LtCpf1 protein sequence, cloning the LtCpf1 protein sequence into a PX330 eukaryotic vector, and constructing a PX330-LtCpf1 plasmid;

(b) the human gene locus CDKN2A is selected to design crRNA, and the design principle is as follows:

1) crRNA includes Spacer (Spacer) and Direct Repeat (DR) sequences in the format: 5 '-direct repeat-crRNA spacer-3' that binds to LtCpf1 protein;

2) the length of the spacer sequence of the crRNA is 15, 17, 19, 21, 23, 25, 27 and 29 base sequences;

3) the length of the direct repetitive sequence of the crRNA is 22 base sequences;

4) the direct repeat sequence of crRNA should contain a stem loop structure (stem loop);

respectively inserting 8 different crRNAs into a target plasmid by a Gibson method, adding a human eukaryotic strong promoter U6 at the upstream of the crRNAs, and constructing 8 different PX330-LtCpf1-crRNA plasmids;

(c) respectively electrotransfering PX330-LbCpf1-crRNA plasmid 2.5ug and 1.5ul ODN of the constructed target human gene locus CDKN2A into HEK293T cells with good states, and collecting all cells after 72h to extract DNA;

(d) performing ODN-PCR by designing a pair of primers near the target gene locus and on the ODN sequence, observing a target band with correct size by agarose gel electrophoresis, and preliminarily identifying that a target cutting event occurs;

(e) designing a proper amplification primer aiming at a target gene sequence, carrying out amplicon high-throughput library construction on DNA, quantifying the Indel rate of a target region, and comparing the gene editing efficiency when the Spacer is respectively 15, 17, 19, 21, 23, 25, 27 and 29 bases;

the amplicon quantitative gene editing efficiency histogram is shown in fig. 9. The results show that LtCpf1 can be effectively guided to target genome editing when the Spacer range is 15-29.

Example 10

In this example, the ability of the V-type CRISPR/Cas12a gene editing system described in example 1 of the present invention to rapidly detect codv-19 nucleic acid in vitro was verified by an in vitro side-cut probe experiment.

(1) Materials: PAM preliminarily identified by LtCpf1 obtained in example 4; the optimal crRNA structure of LtCpf1 obtained in examples 8 and 9; LtCpf1 protein was purified in vitro.

(2) The verification method comprises the following steps: collecting total RNA of a COVID-19 gene expression plasmid vector, amplifying N gene of the new coronavirus at constant temperature by using RT-fluorescent nucleic acid amplification reagent (RAA method), selecting a proper target point according to PAM, combining an in-vitro cutting single-stranded nucleic acid fluorescent probe experiment, observing the capability of LtCpf1 for rapidly detecting COVID-19 nucleic acid at an in-vitro level, and comparing the capability with the capability of LtCpf1 of reference 2 for detecting COVID-19 nucleic acid;

the specific operation is as follows:

(a) extracting total RNA expressed by the COVID-19 plasmid vector in escherichia coli, designing a proper constant-temperature amplification primer, and obtaining an N gene segment of the COVID-19 through an RT-fluorescent nucleic acid amplification reagent (RAA method);

(b) and (3) selecting a target with a better detection effect verified in reference (2) by combining PAM (Polyacrylamide) (TTNA) and LtCpf1, designing and extracorporeally transcribing the crRNA aiming at the N gene of LtCpf1 according to the design principle that the target is combined with the crRNA. (ii) a

(c) Synthesizing a FAM-ssDNA-TAMRA fluorescent probe;

(d) according to Cas12 a: crRNA: mixing the samples with the molar weight of the template being 10:10:1, incubating for 15 minutes at 37 ℃, adding the synthesized single-stranded DNA fluorescent probe, incubating for 1 hour at 37 ℃ in a fluorescent quantitative Polymerase Chain Reaction (PCR) detection system, activating the Cas12 protein to cut the ssDNA sequence in the FAM-ssDNA-TAMRA fluorescent probe when new crown N gene double-stranded DNA exists in the sample, so that the fluorescent group is not inhibited by a quenching group, and recording a curve graph of the change of the fluorescence brightness in a reaction system along with time.

FIG. 10 is a schematic diagram of the result of rapid nucleic acid detection of the COVID-19 gene by the homologous V-type CRISPR/Cas12a gene editing system. A, picture A: we tested different copy numbers of N gene PCR product input and recorded fluorescence-time profiles (6.67 x 10^10, 6.67 x 10^9, 6.67 x 10^8, 6.67 x 10^7, respectively). The results show that LtCpf1 can still generate a rapid and sensitive nucleic acid detection effect when the copy quantity of the N gene is as low as 6.67 x 10^ 7; and B, drawing: fluorescence intensity histograms recorded 60min after the start of the reaction for 4 different N gene copy number experimental groups. The results show that the copy amounts of the 4N genes can effectively activate the activity of the LtCpf1 protein for indiscriminately cutting the ssDNA fluorescent probe.

The reaction conditions of the implementation are the same as that of reference 2, and reference 2 shows that the lower limit of the effective detection of the N gene of the existing V-type CRISPR/Cas12a gene editing system (LbCpf1 protein) is about 10^10 copy numbers, so that the V-type CRISPR/Cas12a gene editing system has higher sensitivity.

Reference (2) is Wang, x, Zhong, m., Liu, y, Ma, p., Dang, l, Meng, q, Wan, w., Ma, x, Liu, j., Yang, g.et al (2020) Rapid and sensitive detection of cove-19 using CRISPR/Cas12a-based detection with naked eye readout, CRISPR/Cas12 a-ner.sci. bright, 65, 1436-.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Sequence listing

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<120> Cpf1 protein and gene editing system

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auauaauuuc uacugaaagu guagaua 27

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uauaauuucu acugaaagug uagaua 26

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auaauuucua cugaaagugu agaua 25

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uaauuucuac ugaaagugua gaua 24

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aauuucuacu gaaaguguag aua 23

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auuucuacug aaaguguaga ua 22

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augcaaacuu uaccgaugau gaaga 25

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<211> 28

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uacgagguug ugaucgaagu ccauaacc 28

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<212> RNA

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uuccuaaaau uacaaauaaa uccug 25

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<211> 26

<212> RNA

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<400> 16

gaugcaguuu ucagauuuug uuuuug 26

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agaaaaguca agauauucaa acuaaa 26

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agguaaaacu ccaaucuggc uug 23

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gccccaauaa uccccacaug uca 23

<210> 21

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<212> RNA

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ucccugucuu cugcaaaggu gag 23

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uucugugugg guucaaacac auu 23

Claims (10)

1. A V-type CRISPR/Cas12a gene editing system, characterized in that: comprising a Cpf1 protein or one or more polynucleotides encoding said Cpf1 protein, and CRISPR RNA or one or more polynucleotides encoding this CRISPR RNA; wherein, the amino acid sequence of the Cpf1 protein is shown as SEQ ID NO.1, or has at least 80% homology with SEQ ID NO.1, preferably has at least 85% homology, preferably has at least 90% homology, further preferably has at least 95% homology, and further preferably has at least 96%, 97%, 98% and 99% homology.

2. The type V CRISPR/Cas12a gene editing system of claim 1, characterized in that: the Cpf1 protein comprises one or more mutant amino acid residues at positions: positions 117, 150, 267, 281, 538, 593, 776.

3. The type V CRISPR/Cas12a gene editing system of claim 1, characterized in that: the Cpf1 protein is a DNA endonuclease, and the Cpf1 protein cleaves double-stranded DNA complementary to CRISPR RNA downstream of the PAM sequence through different nuclease domains; the different nuclease domains are HNH-like nuclease domains or RuvC-like nuclease domains; the PAM sequence is TTNA, wherein N is A, T, C, G.

4. The type V CRISPR/Cas12a gene editing system of claim 1, characterized in that: the CRISPR RNA design principle includes one or more of the following:

1) CRISPR RNA the sequence format is: 5 '-direct repeat binding to Cpf1 protein-spacer complementary to target sequence-3';

2) CRISPR RNA the length of the spacer sequence is 10-30 bases;

3) CRISPR RNA has a direct repeat sequence length of 12-37 bases;

4) CRISPR RNA should contain a stem-loop structure.

5. The type V CRISPR/Cas12a gene editing system according to claim 4, characterized in that: the direct repeat sequence of CRISPR RNA is shown as SEQ ID NO.5, or has at least 80% homology therewith, preferably as shown in any one of SEQ ID NO.6 to 12, or has at least 80% homology with any one of SEQ ID NO.6 to 12.

6. The type V CRISPR/Cas12a gene editing system according to any one of claims 1 to 5, characterized in that: CRISPR RNA is transcribed from a CRISPR Array comprising a direct repeat and a spacer, the spacer of the CRISPR Array comprising a target sequence and an element associated with a Cpf1 protein,

the nucleotide sequence of the element associated with the Cpf1 protein is as shown in SEQ ID NO 13, or has at least 80% homology thereto, and/or,

such as SEQ ID NO 14, or at least 80% homologous thereto, and/or,

such as SEQ ID NO 14, or at least 80% homologous thereto, and/or,

15, or at least 80% homologous thereto, and/or,

such as SEQ ID NO 16, or at least 80% homologous thereto, and/or,

such as SEQ ID NO 17, or at least 80% homologous thereto.

7. The type V CRISPR/Cas12a gene editing system of claim 1, characterized in that: the gene editing system further comprises an accessory protein or one or more polynucleotides encoding the accessory protein; the auxiliary protein comprises one or more of Cas1 protein, Cas2 protein and Cas4 protein,

the Cas1 protein has an amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence at least 80% homologous with the amino acid sequence shown in SEQ ID NO.2, preferably at least 85% homologous, further preferably at least 90% homologous, more preferably at least 95% homologous, and still more preferably at least 96%, 97%, 98%, 99% homologous;

the Cas2 protein has an amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence at least 80% homologous with the amino acid sequence shown in SEQ ID NO.3, preferably at least 85% homologous, further preferably at least 90% homologous, more preferably at least 95% homologous, and still more preferably at least 96%, 97%, 98%, 99% homologous;

the Cas4 protein has an amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence at least 80% homologous with the amino acid sequence shown in SEQ ID NO.4, preferably at least 85% homologous, further preferably at least 90% homologous, more preferably at least 95% homologous, and still more preferably at least 96%, 97%, 98%, 99% homologous.

8. A Cpf1 protein, or a polynucleotide encoding the Cpf1 protein, or a vector comprising the polynucleotide and a vector system comprising one or more polynucleotides encoding the CRISPR RNA, or a complex comprising the Cpf1 protein and the CRISPR RNA protein in the V-type CRISPR/Cas12a gene editing system of any one of claims 1 to 7;

preferably, the polynucleotide is codon optimized for expression in the cell of interest.

9. A gene editing system according to any one of claims 1 to 7, or a Cpf1 protein, polynucleotide, vector system, complex according to claim 8, that binds to or cleaves a structure of DNA function in a biological process; preferably, the structure of the DNA function comprises a crRNA secondary structure, a Cpf1 effector protein domain, or a Cpf1-crRNA complex structure; preferably, the DNA is a prokaryotic or eukaryotic DNA.

10. Use of a gene editing system according to any one of claims 1 to 7, or a Cpf1 protein, polynucleotide, vector system, complex according to claim 8, for editing or detecting a prokaryotic or eukaryotic gene; preferably, it is used for binding, targeted cleavage or non-targeted cleavage of DNA.

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