WO2022199511A1

WO2022199511A1 - Lt1cas13d protein and gene editing system

Info

Publication number: WO2022199511A1
Application number: PCT/CN2022/081945
Authority: WO
Inventors: 田瑞; 谢红娴; 黄龙; 兰凯
Original assignee: 珠海舒桐医疗科技有限公司
Priority date: 2021-03-26
Filing date: 2022-03-21
Publication date: 2022-09-29
Also published as: CN113234702B; CN113234702A

Abstract

Provided is an VI-D CRISPR/Cas13 gene editing system. The system comprises a novel RNA endonuclease, i.e., an Lt1Cas13d protein, or one or more polynucleotides for coding the Lt1Cas13d protein, and CRISPR RNA or one or more polynucleotides for transcribing the CRISPR RNA, the amino acid sequence of the Lt1Cas13d protein being the amino acid sequence as shown in SEQ ID NO. 1, or an amino acid sequence having at least 80% homology with the amino acid sequence as shown in SEQ ID NO. 1.

Description

A Lt1Cas13d protein and gene editing system

technical field

The invention belongs to the technical field of gene editing, in particular to an Lt1Cas13d protein and a gene editing system.

Background technique

Gene editing technology makes it possible to modify the DNA sequence location, such as the first-generation gene editing tools zinc finger nucleases (zinc finger nucleases, ZFNs), the second-generation gene editing tools are similar to small nucleases that activate transcription ( transcription activator-like effector nucleases, TALENs), type II and type V CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR- associated protein) can be used to modify the targeted genome, but these gene editing systems can only target genomic DNA, but cannot target editing of foreign RNA.

Type VI CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR-associated protein) system is an innate immune system from archaea and bacteria. Different from the previous gene editing tools, it uses the principle of nucleic acid base complementary pairing to identify the target RNA sequence and guide the Cas effector protein to perform site-specific cleavage. It has strong applicability, simple design and high efficiency. Among them, the CasRfxCas13d in the VI-D CRISPR/Cas13 system is the CRISPR/Cas system with the highest RNA editing efficiency, and the cleavage of the VI-D CRISPR/Cas13 system has no PFS in both prokaryotic and eukaryotic systems (Protospacer flanking site).

In the huge and various metagenomes, there are hidden microorganisms that have not yet been cultivated or even discovered, and there may be a large number of undiscovered CRISPR/Cas systems, which are used in prokaryotes and eukaryotes, as well as in vitro environments. Activity also needs to be confirmed.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a new and efficient gene editing system for targeted editing of RNA, so as to enrich the existing gene editing tool family.

To achieve the above object, the technical scheme adopted in the present invention is:

A first aspect of the present invention provides an Lt1Cas13d protein and a polynucleotide encoding the above-mentioned Lt1Cas13d protein. The amino acid sequence of the Lt1Cas13d protein is shown in SEQ ID NO.1, or has at least 80% homology with it.

Preferably, the amino acid sequence of the Lt1Cas13d 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 more preferably at least 96%, 97%, 98%, 99% homology.

The Lt1Cas13d protein contains 957 amino acids and is a multi-domain and multifunctional RNA endonuclease. It efficiently cleaves single-stranded RNAs complementary to CRISPR RNAs (crRNAs) through a HEPN-like nuclease domain.

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

A second aspect of the present invention is to provide a vector comprising a polynucleotide encoding the Lt1Cas13d protein.

A third aspect of the present invention is to provide a vector system comprising one or more vectors comprising the above-mentioned polynucleotide encoding the Lt1Cas13d protein, on the same or different vectors One or more polynucleotides comprising transcribed CRISPR RNA.

The fourth aspect of the present invention is to provide a complex and a VI-D type CRISPR/Cas13 gene editing system, the complex comprising the Lt1Cas13d protein and CRISPR RNA. The VI-D type CRISPR/Cas13 gene editing system comprises the Lt1Cas13d protein or one or more polynucleotides encoding the Lt1Cas13d protein, and CRISPR RNA or one or more polynuclei that transcribe the CRISPR RNA Glycosides.

The fifth aspect of the present invention also provides a design principle of CRISPR RNA, including one or more of the following:

1) The length of the spacer sequence of CRISPR RNA is 9-30 base sequences;

2) The spacer sequence of the CRISPR RNA is reverse complementary to the sense strand of the target gene;

3) The direct repeat sequence of CRISPR RNA is 12-36 nucleotides;

4) The direct repeat sequence of CRISPR RNA contains 2 stem-loop structures;

5) The middle of the spacer sequence of CRISPR RNA is the seed region, and no mismatch occurs when it binds to the target sequence.

Preferably, the CRISPR Array is transcribed to obtain a precursor CRISPR RNA (pre-crRNA), the precursor CRISPR RNA is processed and sheared to form the CRISPR RNA, and the CRISPR RNA forms a complex with the Lt1Cas13d protein as a guide RNA,

The CRISPR Array includes a plurality of repeating sequences and spacer sequences, and the spacer sequences of the CRISPR Array include target sequences,

The precursor CRISPR RNA sequence from 5' to 3' is: 5'-direct repeat sequence-spacer sequence-direct repeat sequence-3', and the Lt1Cas13d protein can recognize the direct repeat sequence.

Specifically, the target sequence is DNA reversely transcribed from a short fragment of exogenous RNA or a target sequence designed and artificially synthesized for the target gene.

The spacer sequence in the mature CRISPR RNA transcribed and processed is complementary to the target anchor gene, and guides the Lt1Cas13d protein to cut the gene in the target genome. The mature CRISPR RNA (crRNA) sequence has a direct repeat sequence at the 5' end and a spacer sequence at the 3' end. The mature CRISPR RNA can be used as a guide RNA to form a complex with the Lt1Cas13d protein, and the direct repeat sequence guides the Lt1Cas13d protein and specific RNA targets. On binding, the spacer sequence pairs complementary to the specific RNA target.

According to one embodiment, the repeat sequence of the CRISPR Array is shown in SEQ ID NO: 4, or has at least 80% homology with it. Repeats in the CRISPR Array are transcribed to form direct repeats in the precursor CRISPR RNA.

According to a specific embodiment, when the VI-D type CRISPR/Cas13 gene editing system is used for targeted cleavage of E. coli RNA, the target sequence is as shown in SEQ ID NO: 12;

According to a specific embodiment, when the VI-D type CRISPR/Cas13 gene editing system is used for targeted cleavage of the endogenous gene ANXA4, the target sequence is such as SEQ ID NO: 13 and/or SEQ ID NO :14.

Preferably, the spacer sequence of the CRISPR Array also includes an element related to the Lt1Cas13d protein, and the nucleotide sequence of the element related to the Lt1Cas13d protein is as shown in SEQ ID NO: 5, or has at least 80% identity with it. origin, and/or,

As shown in SEQ ID NO:6, or at least 80% homologous thereto, and/or,

As shown in SEQ ID NO: 7, or at least 80% homologous thereto, and/or,

As shown in SEQ ID NO: 8, or at least 80% homologous thereto, and/or,

As shown in SEQ ID NO: 9, or at least 80% homologous thereto, and/or,

As shown in SEQ ID NO: 10, or at least 80% homologous thereto, and/or,

As shown in SEQ ID NO: 11, or at least 80% homologous thereto.

Preferably, the gene editing system further comprises an accessory protein or one or more polynucleotides encoding the accessory protein.

The accessory proteins help capture foreign genes and participate in precursor CRISPR RNA splicing.

Further preferably, the auxiliary protein includes Cas1 protein and/or Cas2 protein,

Wherein, the Cas1 protein has the amino acid sequence shown in SEQ ID NO: 2 or has at least 80% homology with it, preferably at least 85% homology, more preferably at least 90% homology, More preferably at least 95% homology, still more preferably at least 96%, 97%, 98%, 99% homology;

The Cas2 protein has the amino acid sequence shown in SEQ ID NO:3 or has at least 80% homology with it, preferably at least 85% homology, more preferably at least 90% homology, more preferably At least 95% homology, more preferably at least 96%, 97%, 98%, 99% homology.

The CRISPR RNA (crRNA) of the present invention guides the Lt1Cas13d protein to recognize the invaded foreign genome in the form of base complementary pairing. When bacteria invade by phage or virus, with the assistance of Cas1 protein and Cas2 protein, the DNA reversely transcribed from the short fragment of exogenous RNA is integrated as a new spacer sequence between the direct repeats of the CRISPR Array in the host chromosome. , thereby providing a genetic record of infection. When the body is invaded by foreign genes again, the CRISPR Array is transcribed to produce precursor CRISPR RNA (pre-crRNA), which includes the sequence of the foreign invasion gene. The complementary spacer sequence and the spacer sequence complementary to the element related to the Lt1Cas13d protein are obtained by shearing to obtain a mature CRISPR RNA (crRNA) whose 5' end is a direct repeat sequence and the 3' end is a spacer sequence, and the spacer sequence at the 3' end is A spacer sequence complementary to the sequence of the foreign invasion gene, mature CRISPR RNA (crRNA) acts as a guide RNA (sgRNA) for the Lt1Cas13d protein.

The present invention also provides a structure in which the vector system, the complex, or the VI-D CRISPR/Cas13 gene editing system binds or cuts RNA functions in biological processes; preferably, the The structure that binds or cuts RNA function includes but is not limited to CRISPR RNA (crRNA) secondary structure, Lt1Cas13d effector protein domain or Lt1Cas13d-crRNA complex structure; preferably, the RNA that can be combined or cut by the structure is a prokaryotic organism or eukaryotic RNA.

The Lt1Cas13d protein can recognize and cleave single-stranded RNA complementary to the CRISPR RNA (crRNA) spacer sequence. Unlike the VI-A and VI-B Cas13 systems, the Lt1Cas13d protein recognizes and cleaves single-stranded RNA without PFS (Protospacer flanking site). Therefore, the interference experiment can show that Lt1Cas13d protein has a cleavage effect in prokaryotic system. By artificially designing CRISPR RNA (crRNA) and synthesizing a CRISPR Array containing the target sequence (complementary to the spacer sequence of the designed CRISPR RNA), the VI-D type CRISPR-Cas13 system of the present invention can target almost all the interested genomes RNA sequence.

The sixth aspect of the present invention also provides an application of the VI-D type CRISPR/Cas13 gene editing system in editing prokaryotic or eukaryotic genes.

As a preferred embodiment of the application of the present invention, the VI-D CRISPR/Cas13 gene editing system is used for binding or cleavage at the RNA level.

The invention provides a new VI-D type CRISPR/Cas13 gene editing system, which has new physical and chemical properties and no PFS (Protospacer flanking site) for single-stranded RNA targeted editing.

Compared with the existing CRISPR/Cas13 gene editing system, the VI-D type CRISPR/Cas13 gene editing system of the present invention is more efficient.

Due to the application of the above-mentioned technical solutions, the present invention has the following advantages compared with the prior art:

The present invention finds a new RNA endonuclease, namely Lt1Cas13d protein, and uses it to develop a VI-D CRISPR/Cas13 gene editing system, which is applied to editing prokaryotic or eukaryotic RNA, and is a gene editing system. Toolbox provides new options.

Description of drawings

1 is a schematic diagram of the composition of the VI-D CRISPR/Cas13 gene editing system of Example 1.

Fig. 2 is the RNA secondary structure prediction diagram of the CRISPR RNA (crRNA) molecule recognized by the VI-D type CRISPR/Cas13 gene editing system of the present invention;

Fig. 3 is the interference experiment result of VI-D type CRISPR/Cas13 gene editing system of the present invention;

Figure 4 is a comparative diagram of the relative expression levels obtained by relative quantification of qPCR after the VI-D type CRISPR/Cas13 gene editing system according to the present invention and the VI-D type CasRfxCas13d gene editing system targeted cleavage of endogenous genes.

Detailed ways

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific embodiments.

Example 1

Proteins and related elements related to the type VI CRISPR-Cas13 system were obtained by analyzing, predicting and screening the metagenome to obtain the type VI-D CRISPR/Cas13 gene editing system shown in Figure 1. The VI-D CRISPR-Cas13 gene editing system includes the following components: endonuclease Lt1Cas13d gene, accessory proteins Cas1, Cas2, CRISPR Array. The endonuclease Lt1Cas13d contains 957 amino acids, and its sequence is shown in SEQ ID NO.1; the sequence of the auxiliary protein Cas1 is shown in SEQ ID NO.2, the sequence of Cas2 is shown in SEQ ID NO.3, and the auxiliary protein Cas1 and Cas2 are involved in the capture of exogenous genes and the maturation of CRISPR RNA (crRNA); CRISPR Array includes repeat sequences (the sequence of which is shown in SEQ ID NO. .5 to 11).

With the help of Cas1 protein and Cas2 protein, a short fragment of exogenous RNA is reverse transcribed into DNA or a synthetic sequence (target sequence) is integrated into the repeat sequence of CRISPR Array as a new spacer sequence. CRISPR Array transcription produces precursor CRISPR RNA (pre-crRNA). The sequence of pre-crRNA is from 5' to 3': 5'-direct repeat sequence-spacer sequence-direct repeat sequence-3', the spacer sequence of pre-crRNA includes Sequences complementary to the target sequence and sequences complementary to elements associated with the Lt1Cas13d protein. The pre-crRNA is then sheared 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 sequence or the synthetic sequence (target sequence) is complementary to the sequence, and the direct repeat sequence at the 5' end guides the Lt1Cas13d protein to bind to the target sequence, thereby serving as a guide RNA (sgRNA) to guide the Lt1Cas13d protein to cut the target sequence.

Example 2

This example is to predict the RNA secondary structure of the crRNA molecule recognized by the VI-D CRISPR/Cas13 gene editing system of the present invention.

Because the pre-crRNA is sheared under the action of Lt1Cas13d nuclease to form a mature crRNA with a direct repeat sequence at the 5' end and a spacer sequence at the 3' end, as a guide RNA (guide RNA), the spacer sequence at the 3' end is the same as the target gene. The sense strands are complementary to each other, so secondary structure can be predicted from the direct repeats retained at the 5' end.

(1) Material: repeat sequence (SEQ ID NO: 4),

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

(3) Prediction method: The secondary structure formed by the repeat sequence in vitro at 37°C was simulated by using the online application NUPACK, as shown in Figure 2.

It can be seen from Figure 2 that the repeat sequence has two stem-loop structures, so the RNA secondary structure of the crRNA molecule recognized by the VI-D CRISPR/Cas13 gene editing system of the present invention has two stem-loop structures.

Example 3

In this example, the cleavage ability of the VI-D CRISPR/Cas13 gene editing system of the present invention at the prokaryotic level was determined by interference experiments. The results of the interference experiment are shown in Figure 3.

(1) Materials: VI-D type CRISPR/Cas13 gene editing system-related genes predicted in Example 1.

(2) Verification method: In this example, a prokaryotic verification system was constructed for the VI-D CRISPR/Cas13 gene editing system of the present invention to verify its cutting effect.

The specific operations are as follows:

(a) Insert the newly discovered VI-D CRISPR/Cas13 gene editing system (including the endonuclease Lt1Cas13d gene and its corresponding CRISPR Array sequence) of the present invention into the pET28a vector, and the Lt1Cas13d gene sequence is encoded by Escherichia coli Sub-optimization, adding artificially synthesized gene sequence to CRISPR Array, the sequence is shown in SEQ ID NO: 12, and adding strong heterologous promoter J23119 to the upstream of Cas13d gene and CRISPR Array to construct prokaryotic expression pET28a-Cas13d plasmid;

(b) insert the artificially synthesized target sequence corresponding to the artificially synthesized gene sequence SEQ ID NO: 12 in the CRISPR Array after the first initiation codon of the chloramphenicol gene of the pACYC184 plasmid to construct the pACYC184-target plasmid;

(c) Co-electroporation containing pET28a-Cas13d and pACYC184-target into Escherichia coli DH5a, co-electroporating pET28a-Cas13d and empty pACYC184 into Escherichia coli DH5a as a control, after recovery at 37°C for 1 h, the bacterial solution was diluted according to gradient And drop in the SOB medium containing kanamycin (50ug/ml) and chloramphenicol (30ug/ml) double resistance to incubate at 37°C for 12-16h, and observe the number of monoclonal colonies under different concentration gradients.

Figure 3 shows that Lt1Cas13d can effectively target and cleave RNA sequences in E. coli by interference experiments. The right column of Figure 3 is the targeted cleavage target plasmid of Lt1Cas13d, and the left column of Figure 3 is the single Lt1Cas13d and empty pACYC184. The number of monoclonal colonies was observed by gradient dilution. Efficiently targeted cleavage of RNA sequences.

Example 4

This example compares the effects of the VI-D CRISPR/Cas13 gene editing system of the present invention and the VI-D CasRfxCas13d gene editing system at the eukaryotic level by targeting the eukaryotic endogenous gene ANXA4 and qPCR relative quantification. cutting ability. The relative quantification results of qPCR are shown in Figure 4.

(1) Materials: The VI-D CRISPR/Cas13 gene editing system-related genes predicted in Example 1 and the discovered VI-D CRISPR gene CasRfxCas13d (referred to as CasRx, which has the strongest cutting effect), are disclosed in the literature 1. Konermann, Silvana , et al. "Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors." Cell (2018) pii: S0092-8674(18) 30207-1.2. Yan, Winston X., et al. "Cas13d Is a Compact RNA -Targeting Type VI CRISPR Effector Positively Modulated by a WYL-Domain-Containing Accessory Protein."Mol Cell.(2018)pii:S1097-2765(18)30173-4.) and its related genes.

(2) Verification method: In this example, a eukaryotic verification system is constructed for the VI-D CRISPR/Cas13 gene editing system of the present invention to verify its cutting effect and compare its cutting effect with CasRx;

The specific operations are as follows:

The CRISPR RNA (crRNA) targeting the endogenous gene ANXA4 was designed according to the following principles:

a) The length of the spacer sequence of crRNA is 30 base sequences;

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

c) The direct repeat sequence of crRNA is a 36-base sequence;

d) The direct repeat sequence of crRNA should contain 2 stem loop structures;

e) There is a seed region in the middle of the spacer sequence of the crRNA, and no mismatch can occur when it binds to the target sequence.

The crRNA is obtained by shearing and processing the precursor crRNA transcribed by CRISPR Array. The precursor crRNA includes spacer (spacer) and direct repeat sequence (Direct Repeat, DR). The sequence from the 5th end to the 3rd end is: 5' direct repeat sequence-spacer Sequence-Direct Repeat Sequence-3'.

Two targets, crRNA-1, crRNA-2, and Nontarget (NT) that do not target ANXA4 were designed on the ANXA4 exon. The lengths of crRNA-1, crRNA-2 and Nontarget (NT) are all 30 bp. The sequences are SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15:

(3) In the minicircle vector (minicircle vector has been described in the literature [1] Darquet AM, Rangara R, Kreiss P, Schwartz B, Naimi S, Delaère P, Crouzet J, Scherman D. Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer.Gene Ther.1999Feb;6(2):209-18.doi:10.1038/sj.gt.3300816.PMID:10435105.[2]Chen ZY,He CY,Ehrhardt A,Kay MA.Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo.Mol Ther.2003Sep;8(3):495-500.doi:10.1016/s1525-0016(03)00168-0.PMID:12946323.[3 ]Kay MA,He CY,Chen ZY.A robust system for production of minicircle DNA vectors.Nat Biotechnol.2010Dec;28(12):1287-9.doi:10.1038/nbt.1708.Epub 2010Nov 21.PMID:21102455; Insert the VI-D CRISPR/Cas13 gene editing system of the present invention (including the endonuclease Lt1Cas13d protein gene and its corresponding CRISPR array sequence) or VI-D CRISPR gene CasRfxCas13d (including CasRx) into PMCID: PMC4144359. protein gene and its corresponding CRISPR array sequence), Lt1Cas13d protein gene and CasRx protein gene sequence were optimized for human origin, and CRISPR arrays with crRNA-1, crRNA-2 and Nontarget (NT) sequences were synthesized respectively. The promoter EF-1a was added upstream of the Lt1Cas13d protein gene, and the human promoter U6 was added upstream of the CRISPR array to construct eukaryotic expression minicircle-Cas13d-crRNA and minicircle-CasRx-crRNA plasmids, including the spacer sequence targeting the endogenous gene ANXA4 The minicircle-Cas13d-crRNA-1, minicircle-Cas13d-crRNA-2, minicircle-CasRx-crRNA-1, minicircle-CasRx-crRNA-2 plasmids and plasmids that do not target any endogenous gene minicircle-CasRx-crRNA- NT, minicircle-CasRx-crRNA-NT;

(4) The above-constructed minicircle-Cas13d-crRNA and minicircle-CasRx-crRNA plasmids were transferred into blank HEK293 cells by Hp transfection to transfer into minicircle-Cas13d-crRNA-1, minicircle-Cas13d-crRNA -2 plasmid and minicircle-CasRx-crRNA-1 and minicircle-CasRx-crRNA-2 were used as the experimental group, and the plasmids transferred into minicircle-Cas13d-crRNA-NT and minicircle-CasRx-crRNA-NT were used as the negative control group. The HEK293 cells in the untransfected group were blank control. After culturing for 48 hours, the cells were harvested, RNA was extracted, and reverse transcribed into cDNA, and GAPDH was used as the internal reference gene for qPCR relative quantitative analysis. The target endogenous gene ANXA4 in the experimental group was observed. Whether the expression level decreased relative to the blank control group.

It can be seen from Figure 4 that the expression levels of the target endogenous gene ANXA4 in the experimental groups of VI-D type CRISPR/Cas13 and VI-D type CasRfxCas13d were significantly lower than those in the respective blank control groups, and the expression levels of the VI-D type CRISPR/Cas13 target genes were significantly reduced. The degree of decline is more obvious than that of VI-D CasRfxCas13d. It is proved that the VI-D type CRISPR/Cas13 gene editing system in the present invention has a targeted cutting effect in eukaryotes, and the cutting effect is stronger than the existing VI-D type CasRfxCas13d.

The above-mentioned embodiments are only intended to illustrate the technical concept and characteristics of the present invention, and the purpose thereof is to enable those who are familiar with the art to understand the content of the present invention and implement them accordingly, and cannot limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included within the protection scope of the present invention.

Claims

A VI-D type CRISPR/Cas13 gene editing system, characterized in that: it comprises Lt1Cas13d protein or one or more polynucleotides encoding the Lt1Cas13d protein, and CRISPR RNA or one or more of transcribing the CRISPR RNA. A variety of polynucleotides, the amino acid sequence of the Lt1Cas13d protein is the amino acid sequence shown in SEQ ID NO.1 or the amino acid sequence with at least 80% homology with it.
The VI-D type CRISPR/Cas13 gene editing system according to claim 1, wherein the amino acid sequence of the Lt1Cas13d protein has at least 85% homology with SEQ ID NO.1, preferably at least 90% homology, more preferably at least 95% homology, still more preferably at least 96%, 97%, 98%, 99% homology.
The VI-D type CRISPR/Cas13 gene editing system according to claim 1, wherein the Lt1Cas13d protein is an RNA endonuclease, and the Lt1Cas13d protein is complementary to CRISPR RNA through HEPN-like nuclease domain cleavage of single-stranded RNA.
The VI-D type CRISPR/Cas13 gene editing system according to claim 1, wherein the design principles of the CRISPR RNA include one or more of the following:

1) The length of the spacer sequence of CRISPR RNA is 9-30 base sequences;

2) The spacer sequence of the CRISPR RNA is reverse complementary to the sense strand of the target gene;

3) The direct repeat sequence of CRISPR RNA is 12-36 nucleotides;

4) The direct repeat sequence of CRISPR RNA contains 2 stem-loop structures;

5) The middle of the spacer sequence of CRISPR RNA is the seed region, and no mismatch occurs when it binds to the target sequence.
The VI-D type CRISPR/Cas13 gene editing system according to any one of claims 1 to 4, wherein the CRISPR Array is transcribed to obtain a precursor CRISPR RNA, and the precursor CRISPR RNA is cut and processed to form the CRISPR RNA , the CRISPR RNA forms a complex with the Lt1Cas13d protein as a guide RNA,

The CRISPR Array includes a plurality of repeating sequences and spacer sequences, and the spacer sequences of the CRISPR Array include target sequences,

The precursor CRISPR RNA sequence from 5' to 3' is: 5'-direct repeat sequence-spacer sequence-direct repeat sequence-3', and the Lt1Cas13d protein can recognize the direct repeat sequence.
The VI-D type CRISPR/Cas13 gene editing system according to claim 5, wherein the repeat sequence of the CRISPR Array is as shown in SEQ ID NO: 4, or has at least 80% homology with it.
The VI-D type CRISPR/Cas13 gene editing system according to claim 5, wherein the spacer sequence of the CRISPR Array further comprises an element related to the Lt1Cas13d protein,

The nucleotide sequence of the element related to the Lt1Cas13d protein is shown in SEQ ID NO: 5, or has at least 80% homology therewith, and/or,

As shown in SEQ ID NO:6, or at least 80% homologous thereto, and/or,

As shown in SEQ ID NO: 7, or at least 80% homologous thereto, and/or,

As shown in SEQ ID NO: 8, or at least 80% homologous thereto, and/or,

As shown in SEQ ID NO: 9, or at least 80% homologous thereto, and/or,

As shown in SEQ ID NO: 10, or at least 80% homologous thereto, and/or,

As shown in SEQ ID NO: 11, or at least 80% homologous thereto.
The VI-D type CRISPR/Cas13 gene editing system according to any one of claims 1 to 7, wherein the gene editing system further comprises an accessory protein or one or more encoding the accessory protein. A kind of nucleotide sequence; Described accessory protein includes Cas1 protein and/or Cas2 protein,

The Cas1 protein has the amino acid sequence shown in SEQ ID NO: 2 or has at least 80% homology with it, preferably at least 85% homology, more preferably at least 90% homology, more preferably have at least 95% homology, preferably at least 96%, 97%, 98%, 99% homology;

The Cas2 protein has the amino acid sequence shown in SEQ ID NO:3 or has at least 80% homology with it, preferably at least 85% homology, more preferably at least 90% homology, more preferably At least 95% homology, more preferably at least 96%, 97%, 98%, 99% homology.
A Lt1Cas13d protein as described in the VI-D type CRISPR/Cas13 gene editing system according to any one of claims 1 to 8, or a polynucleotide encoding the Lt1Cas13d protein, or comprising the polynucleotide A vector, or a vector system comprising the polynucleotide and one or more polynucleotides encoding the CRISPR RNA, or a complex comprising the Lt1Cas13d protein and the CRISPR RNA.
The protein, polynucleotide, vector, vector system or complex of claim 9, wherein the nucleotide is codon-optimized for expression in a target cell.
A VI-D type CRISPR/Cas13 gene editing system according to any one of claims 1 to 8, or the Lt1Cas13d protein according to claim 9, a polynucleotide encoding the Lt1Cas13d protein, a carrier, a carrier system , the structure of complexes that bind or cleave RNA functions during biological processes.
The structure for binding or cutting RNA function according to claim 11, wherein the structure for binding or cutting RNA function comprises CRISPR RNA secondary structure, Lt1Cas13d effector protein functional domain or Lt1Cas13d-CRISPR RNA complex structure.
The structure for binding or cleaving RNA function according to claim 11, wherein the RNA capable of being bound or cleaved by the structure is a prokaryotic or eukaryotic RNA.
A VI-D type CRISPR/Cas13 gene editing system according to any one of claims 1 to 8, or the Lt1Cas13d protein according to claim 9, a polynucleotide encoding the Lt1Cas13d protein, a carrier, a carrier system , the application of complexes in editing prokaryotic or eukaryotic RNA.