CN113528515B

CN113528515B - CRISPR-Cas13a based technology for interfering and blocking reverse transcription transposition of viruses

Info

Publication number: CN113528515B
Application number: CN202010321800.9A
Authority: CN
Inventors: 李轩; 荆新云; 张牛冰; 朱艳
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2020-04-22
Filing date: 2020-04-22
Publication date: 2023-05-05
Anticipated expiration: 2040-04-22
Also published as: CN113528515A

Abstract

The invention provides a technology for blocking reverse transcription transposition of a reverse transcription organism based on CRISPR-Cas13a intervention. The invention discloses a technology for blocking reverse transcription and transposition of a reverse transcription organism based on CRISPR-Cas13a intervention, a system for blocking reverse transcription and transposition of the reverse transcription organism in eukaryotic cells and application thereof. In the present invention, a model system of Tf1 retrovirus was studied and demonstrated. The technical scheme of the invention not only realizes the inhibition of the reverse transcription and transposition by using the Cas13a for the first time, but also has ideal efficiency of blocking the reverse transcription and transposition.

Description

CRISPR-Cas13a based technology for interfering and blocking reverse transcription transposition of viruses

Technical Field

The invention belongs to the field of virology, and in particular relates to a technology for blocking reverse transcription and transposition of viruses based on CRISPR-Cas13a intervention.

Background

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a regularly clustered, intermittent short palindromic repeat, an adaptive immunization modality in most bacteria and archaea. In recent years, many molecular tools for knocking out genes or introducing mutations inside genes using CRISPR systems have been successfully developed, but tools capable of knocking out genes or editing genes at the RNA level are also urgently needed.

CRISPR has been used as a tool for studying inactivation of endogenous retroviruses against exogenous retroviral pathogens and in organ transplants. CRISPR-Cas9 is a system designed to combat animal and plant retroviral pathogens. CRISPR-Cas9 can effectively inactivate potential HIV-1 in infected cells by targeting HIV-1 essential genes or LTRs, can target cytokines or HIV-1 genome, reduce HIV-1 infection and eliminate provirus. Recently studied CRISPR-Cas9 inactivated Endogenous Retroviruses (ERVs) in pigs to create ERV inactivated animals for xenografts.

However, as research continues, CRISPR-Cas9 exposes its drawbacks and limitations, such as poor inhibition, severe off-target effects, etc. In practical applications, the binding of CRISPR-Cas9 to the target is not precise, followed by a cleavage operation after recognition of the sequence. For example, attempts by chinese scientists to edit human embryos have found many nonspecific cuts, but have resulted in many mutations. Therefore, scientists are looking for new and more efficient gene editing systems while improving Cas9 protein accuracy.

In summary, in the art, although there have been many applications for gene editing using Cas9, further research and exploration is urgently needed to develop more effective antiviral drugs in terms of viral inhibition, particularly in terms of inhibiting retroviruses such as HIV and the like. Furthermore, there is also a lack of test models in the art for detecting the intracellular translocation of reverse transcribed organisms.

Disclosure of Invention

The invention aims to provide a technology for blocking reverse transcription and transposition of viruses based on CRISPR-Cas13a intervention.

In a first aspect of the invention, a system for blocking retrotransposition of a retroactive organism in a eukaryotic cell is provided, comprising a Cas13a expression cassette and a crRNA expression cassette; wherein the crRNA targets an essential gene, a Long Terminal Repeat (LTR), or a region adjacent thereto of the reverse transcribed organism.

In a preferred embodiment, the essential gene comprises a gene selected from the group consisting of: structural genes, regulatory genes.

In another preferred embodiment, the structural or regulatory genes include (but are not limited to): gag, PR, RT, IN, env, tat, rev, vpu, nef, vpr or Vpx.

In another preferred embodiment, in the system, the Cas13a expression cassette and the crRNA expression cassette are located in the same or different expression construct (expression vector); preferably, the two expression cassettes are located in different expression constructs, the Cas13a expression cassette is integrated into the genome of the eukaryotic cell in which the crRNA expression cassette is expressed episomally.

In another preferred embodiment, said blocking reverse transcription transposition of a reverse transcription organism in a eukaryotic cell comprises: reverse transcribed organisms are prevented from replication and jumping in eukaryotic cells by a reverse transcription step.

In another preferred embodiment, the reverse transcription organism comprises: retroviruses (retroviruses), mimics of retroviruses; preferably, the mimetic of the retrovirus is retrotransposon Tf1.

In another preferred example, the retrotransposon Tf1 comprises: a retrotransposition element for detecting a screening gene for retrotransposition events.

In another preferred embodiment, the reverse seat element comprises an operatively linked gene selected from the group consisting of: long Terminal Repeats (LTRs), gag, PR, RT, IN; the long terminal repeat includes a 5'LTR and a 3' LTR.

In another preferred embodiment, the screening gene is a resistance screening gene, a Marker gene or a reporter gene; preferably, the resistance gene is Neo.

In another preferred embodiment, the marker genes include, but are not limited to: nat (nourseothricin, nociceptin gene).

In another preferred embodiment, the reporter gene includes, but is not limited to: fluorescent proteins such as GFP, mCherry, luciferase, etc.

In another preferred embodiment, the screening gene is located upstream of the 3' LTR.

In another preferred embodiment, the screening gene is located adjacent to the long terminal repeat.

In another preferred embodiment, said retroviral mimetic further comprises a promoter operably linked to said retrotransposon that drives transposition of said retrotransposon; preferably, the promoter is a regulatable promoter; more preferably, the regulatable promoter is an nmt1 promoter, which is inhibited by thiamine.

In another preferred embodiment, the crRNA expression cassette comprises the following elements operably linked: direct Repeat (DR), gRNA; preferably, the crRNA expression cassette comprises the following elements operably linked: promoters, direct Repeat (DR), gRNA and ribozymes (HDVR); preferably, the promoter is a rrk1 promoter.

In another preferred embodiment, the nucleotide sequence of the direct repeat is shown as SEQ ID NO. 11, which fully binds to the features of Cas13a via the present inventors' design according to the present invention. The direct repeat sequence is connected with the gRNA and is used as an element of a crRNA expression cassette, so that the Cas13a protein and the crRNA are well combined, and the targeted cutting effect of the Cas13a/crRNA is facilitated.

In another preferred embodiment, the structural or regulatory gene comprises Gag and the crRNA targets position 306 or 360 of the Gag gene; more preferably, the gRNA is selected from: nucleotide sequences at positions 4-31 in the sequence shown in SEQ ID NO. 3 or 5, or their reverse complements.

In another preferred embodiment, the Cas13a is Cas13a (LshCas 13 a) derived from ciliated bacteria (Leptotrichia shahii); preferably, when the eukaryotic cell is a yeast, the Cas13a expression cassette is integrated at the yeast chromosome II LEU1 site.

In another aspect of the invention, there is provided the use of a system as described in any of the preceding to prepare an agent (including scientific agents) or composition (including pharmaceutical compositions) that blocks retrotransposition of a virus in eukaryotic cells.

In a further aspect of the invention there is provided the use of a system as described in any of the preceding claims for performing an experimental test of transposition of a reverse transcribed organism in a eukaryotic cell or for preparing an experimental test system.

In another aspect of the invention, there is provided a method of blocking retrotransposition of a retroorganism in a eukaryotic cell comprising: (1) providing a system as described in any of the preceding; (2) Introducing the system of (1) into a eukaryotic cell, whereby the eukaryotic cell has the ability to block retrotransposition of a retroactive organism.

In a preferred embodiment, the eukaryotic cell is a yeast cell, a plant cell, or an animal cell; preferably, the yeast cells comprise schizosaccharomyces cells.

In another aspect of the invention, there is provided a kit for blocking retrotransposition of a retroactive organism in a eukaryotic cell comprising any of the systems described above.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, a flow chart of the construction of pDIAL-HFF 1-LshCAs13a plasmid.

FIG. 2, flow chart of construction of psk- (LshCAs 13a gRNA) -Tf1 plasmid.

FIG. 3, flow chart of construction of pHL414- (Lshcas 13a gRNA) -Tf1 plasmid.

FIG. 4, technique for interfering with transposition based on CRISPR-Cas13a targeted reverse seat transposition Tf1 RNA intermediate, statistics of transposition efficiency. Wherein, the upper panel is a schematic diagram of an expression system that blocks viral retrotransposition based on CRISPR-Cas13a intervention; the lower panels show the colony formation assay results for Tf1-835 and Tf 1-1165.

Detailed Description

In view of the current situation that the prior art is not successful in inhibiting viruses, particularly inhibiting retroviruses, the inventor of the invention has intensively studied and disclosed a technology for blocking retrotransposition of a retroactive organism based on CRISPR-Cas13a intervention, a system for blocking retrotransposition of a retroactive organism in eukaryotic cells and application thereof. The technical scheme of the invention not only realizes the inhibition of the reverse transcription and transposition by using the Cas13a for the first time, but also has ideal efficiency of blocking the reverse transcription and transposition.

Terminology

As used herein, the term "Retrotransposon" refers to a transposon that is RNA mediated transposition, which is accomplished by transcription of mRNA, followed by reverse transcription into a new element that is integrated into the genome to accomplish the transposition. Retroviruses and transposons replicate and jump in eukaryotic cells through a reverse transcription step. The term "reverse transcription" may also be referred to as "reverse transcription".

As used herein, a "reverse transcribed organism" refers to an organism, including biological cells, that undergoes reverse transcription and transposition. In some preferred embodiments of the invention, the "retrovirus organism" is a retrovirus or a mimic of a retrovirus. Unless otherwise indicated, the "mimetibody of a retrovirus" is an LTR-type retrotransposon, which has a structure and transposition pattern similar to that of a retrovirus. In a more specific mode of the invention, the mimetic of the retrovirus is retrotransposon Tf1.

As used herein, the "guide RNA (crRNA) sequence" is a sequence that is reverse-complementary to the targeted sequence of the site being edited.

As used herein, the "crRNA" is a functional molecule that performs a targeted cleavage function; preferably, its functioning requires two parts: (1) A gRNA sequence portion and (2) a DR sequence portion that interacts with Cas13a, both of which constitute a crRNA expression cassette; DR allows Cas13a protein to bind well to crRNA.

As used herein, the "screening gene for detecting a retrotransposition event" refers to a gene that has a reporter function for the occurrence or absence of a retrotransposition event or can be detected manually. For example, it may be some resistance screening gene or a gene that appears colored or fluorescent upon its expression.

By "operably linked" or "operably linked" is meant a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed in a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, whereby the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, the term "element" refers to a series of functional nucleic acid sequences useful for expression of a protein, where the term "element" is systematically constructed to form an expression construct. The sequences of the "elements" may be those provided in the present invention, and include variants thereof, as long as the variants substantially retain the function of the "elements" obtained by inserting or deleting some bases (e.g., 1 to 50bp; preferably 1 to 30bp, more preferably 1 to 20bp, still more preferably 1 to 10 bp), or performing random or site-directed mutation, etc.

As used herein, the term "expression cassette" refers to a gene expression system comprising all the necessary elements necessary for expression of a protein of interest, typically including the following elements: promoters, gene sequences encoding proteins, terminators. In addition, resistance elements, screening (selection) elements or reporter elements may optionally be included, which are operatively linked.

As used herein, a "retrovirus" is a virus that undergoes retrotransposition. For example, the retroviruses include (but are not limited to): retroviruses include (but are not limited to): human Immunodeficiency Virus (HIV), endogenous Retroviruses (ERVs), human T Lymphocyte Virus (HTLV), and the like.

As used herein, the term "blocking" includes "inhibition", "blocking", and the like.

System for blocking reverse transcription and transposition and application thereof

In the present invention, a system is established based on CRISPR-Cas13a to interfere with blocking of retrotransposition of viruses in eukaryotic cells, including yeast cells. The Cas13a has a function of targeting single-stranded RNA (ssRNA). Cas13a may bind to a specific targeted ssRNA under the guidance of the crRNA and cleave the ssRNA. In a preferred form of the invention, using the LshCAs13a protein from ciliated bacteria (Leptotrichia shahii), the inventors have found that it has a precise editing function when applied to the system of the invention.

The system for blocking reverse transcription and transposition of a reverse transcription organism in a eukaryotic cell comprises a Cas13a expression cassette and a crRNA expression cassette.

Although the Cas13a protein and the Cas9 protein belong to the CRISPR family, the protein structures of the Cas13a protein and the Cas9 protein are different, and the molecular weights of the Cas13a protein and the Cas9 protein are different; meanwhile, the action mechanisms of the two are also quite different. In view of the great difference between the two, the gene editing principle, intracellular working mode and editing result are also obvious, and the two cannot be mutually used or effect deduction can not be carried out.

In a preferred embodiment of the invention, a subspecies of Cas13a protein is used, namely LshCas13a derived from ciliated bacteria (Leptotrichia shahii), the amino acid sequence of which may be as shown in GenBank accession number wp_018451595.1, or a conservatively mutated polypeptide thereof. By "conservatively modified polypeptide" is meant a polypeptide that retains essentially the same biological function or activity as the polypeptide. The "conservatively modified polypeptide" may be (i) a polypeptide having one or more (e.g., 1-50, 1-30, 1-20, 1-10 or 1-5) conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence or sequence used to purify the polypeptide or a proprotein sequence, or fusion protein with the formation of an antigen IgG fragment). Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

In the present invention, the crRNA targets a number of gene targets, including: essential genes, long Terminal Repeats (LTRs), etc., of the reverse transcribed organism, or adjacent regions thereof.

The essential genes of the reverse transcription organism are, for example but not limited to, their structural genes, their regulatory genes, etc. Such as, but not limited to: gag, PR, RT, IN, env, tat, rev, vpu, nef, vpr, vpx, etc. The essential gene may be different depending on the kind of the reverse transcription organism. It is preferable to target genes located between two LTRs, more particularly genes located near the LTRs. For example, in embodiments of the invention, the targeted gene is placed at a location between the essential gene and the LTR.

The Cas13a expression cassette and crRNA expression cassette may be located in the same expression construct (expression vector) or in different expression constructs. As a preferred mode of the invention, in the system, the two expression cassettes are located in different expression constructs, the Cas13a expression cassette is integrated into the genome of the eukaryotic cell for expression, and the crRNA expression cassette is expressed free in the eukaryotic cell. This arrangement facilitates stable expression and accurately blocks reverse transcription transposition of reverse transcribed organisms within eukaryotic cells.

The system for blocking reverse transcription and transposition can effectively prevent reverse transcription organisms (retroviruses or transposons) from replicating and jumping in eukaryotic cells through a reverse transcription step. In particular embodiments of the invention, the prevention of reverse transcription of a stopped virus/transposon by inducing simultaneous expression of Cas13a and crRNA is achieved, and high efficiency of suppressing reverse transcription and transposition is obtained.

The system for blocking reverse transcription and transposition can be prepared into a kit for blocking reverse transcription and transposition of a reverse transcription organism in eukaryotic cells, thereby being convenient for people to use. Preferably, the kit may further comprise instructions for use and the like.

Test system for blocking reverse transcription transposition and application thereof

In order to accurately understand the blocking effect of the system on reverse transcription, the inventor also establishes a test system for blocking reverse transcription transposition after intensive research.

The test system blocking retrotransposon contains retrotransposon Tf1.Tf1 is a retrotransposon from Schizosaccharomyces cerevisiae (Schizosaccharomyces pombe), encoding a long multimeric protein flanked by 385 base Long Terminal Repeats (LTRs). LTR retrotransposons and retroviruses share many common features in terms of genomic structure, replication machinery and life cycle. Both LTR retrotransposons and retroviruses replicate by reverse transcription and propagate by integration into the host genome, which is dependent on the host transcription and translation machinery. Both Tf1 and retroviruses use RNA as an intermediate to synthesize mRNA by transcription, and then reverse transcription to synthesize new elements for integration into the genome to accomplish interference with the host genome. Thus, LTR retrotransposons can be used as an effective model for the study of retroviruses. In a preferred mode of the invention, a plasmid pHL414 expressing Tf1 in schizosaccharomyces cells is selected and modified.

As a preferred mode of the invention, the Tf1 comprises a retrotransposable element (LTR, gag, PR, RT, IN may be included at both ends) and a screening gene for detecting retrotranspositions. In some embodiments, the transcriptional direction of the screening gene may be opposite to the retrotransposon; preferably, the crRNA is targeted to cleave Gag or other essential elements; when Cas13a and crRNA are both expressed, the cells expressing the selected gene are reverse transcription-transposed cells, with the remainder being reverse transcription-transposed-inhibited cells.

In a more preferred manner, the promoter driving expression of the reverse seat element is regulatable (e.g., inducible or repressible). Using this system, the inventors can controllably regulate the expression of a retrotransposable system (e.g., using a regulatable promoter such as nmt1, which is inhibited by thiamine, and which can be expressed when the thiamine is removed), with retrotranspositions occurring; simultaneously, cas13a and crRNA were expressed and the change in retrotransposition was observed.

The test system of the present invention may be used to perform a test in eukaryotic cells (e.g., yeast cells). In a specific embodiment of the invention, a technology for blocking retrotransposition of viruses/transposons based on CRISPR-Cas13a intervention is provided by utilizing schizosaccharomyces as a platform for technical development, and the specific operation method is as follows:

(1) Expression of CRISPR-Cas13a proteins

The invention selects a class VI CRISPR effect protein Cas13a, wherein the Cas13a has the functions of combining and cutting ssRNA. The invention constructs a carrier for expressing LshCAs13a protein. In a preferred embodiment of the invention, the expression vector established is pDIAL-HFF 1-LshCAs13a. Thereafter, the present inventors linearized and transformed the plasmid into schizosaccharomyces, and inserted an LshCas13a protein expression cassette into the LEU1 site of the yeast chromosome II using homologous recombination, thereby generating a yeast strain expressing a single copy of LshCas13a gene in the yeast genome. Schizosaccharomyces was cultivated using MM medium to express LshCas13a protein.

(2) Construction of crRNA and reverse seat model System of CRISPR-Cas13a cleavage RNA target

The invention selects a schizosaccharomyces triple promoter rrk1, and the DR sequence of CRISPR-Cas13a is behind the promoter, so that the Cas13a protein is combined with crRNA; after the DR sequence, a gRNA spacer sequence is inserted (place holder with BspQI), and gRNA can be connected to BspQI restriction sites; following the gRNA insertion site, a ribozyme (HDVR) that cleaves its 5' end; the design can produce a gRNA with an accurate sequence.

Constructing an intermediate vector pSK- (Lshcas 13a gRNA) -target for expressing gRNA, which specifically comprises the following steps: cloning the expression cassette of crRNA onto the expression plasmid pHL414 vector of retrotransposon Tf1 results in pHL414- (LshCas 13a gRNA) -target plasmid.

The pHL414- (LshCas 13a gRNA) -target plasmid was then transformed into a schizosaccharomyces-derived strain (containing the integrally expressed LshCas13a protein). By inducing simultaneous expression of Cas13a and crRNA, the intermediate RNA is cleaved, interfering with the retrotransposition of the arrested virus/transposon.

Using the established retrotransposon model system, the inventors quantitatively determined the transposition efficiency of retrotransposon Tf1, and learned the interference efficiency of CRISPR-Cas13a on Tf1 transposition activity by targeting Tf1 RNA intermediates, and determined the situation where retrotransposition was blocked (inhibited).

The test system for blocking reverse transcription and transposition can be prepared into a kit for blocking reverse transcription and transposition of a reverse transcription organism in eukaryotic cells, thereby being convenient for people to use. Preferably, the kit may further comprise instructions for use and the like.

The CRISPR-Cas13a based RNA editing technique of the present invention suppresses the retrotransposable activity of the virus/transposon. The technology can also be used for operation and interference blocking of transposition and infection amplification of retrovirus, and provides an effective means for interference treatment of retrovirus (such as HIV) diseases.

The invention has the positive progress effects that:

(1) The invention provides a new action mechanism for the interference of retrovirus pathogens by utilizing CRISPR-Cas13a technology for the first time, and targets genes are disabled by targeting RNA intermediates (RNA transcripts in the reverse transcription process). There is no precedent in the art to successfully intervene in the retrotransposition of a stopped virus/transposon using CRISPR-Cas13 a.

(2) In the invention, a CRISPR-Cas13 a-based DNA editing technology constructs and implements a CRISPR-Cas13a gene editing system in eukaryotic cells (schizoyeast is an example) for the first time, and has a remarkable effect on the reverse seat efficiency of viruses/transposons for interference prevention.

(3) The CRISPR-Cas13a based DNA editing technique of the present invention significantly inhibits the transposition activity of eukaryotic (schizo-yeast, for example) viruses/retrotransposons. Meanwhile, the technology can also be used for operating and interfering with the research of retrovirus, and provides an effective tool for the research of RNA virus.

(4) The technical scheme of the invention has simple operation method, is superior to similar tools such as CRISPR-Cas9 and the like, and has higher blocking efficiency.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Materials and methods

pC001 plasmid: see Abudayyeh, O.O. et al C2c2 is a single-component programmable RNA-guide RNA-targeting CRISPR effector.science 353, aaf5573 (2016).

psk- (LshCas 13a gRNA) backbone plasmid: see, jin, X.et al, completion of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA coding, nucleic Acids Res.46,90 (2018). In the framework plasmid, the DR sequence is: ccaccccaatatcgaaggggactaaaac (SEQ ID NO: 11). The inventors found that the DR sequence is linked to the gRNA, well suited for allowing good binding of Cas13a protein to crRNA, facilitating targeted cleavage of Cas13 a/crRNA.

pHL414-2 plasmid: see Garcia-merez, j.l. fransposons and retrotrans posons (Humana Press, 2016).

Example 1 expression of CRISPR-Cas13a proteins in eukaryotic cells

The establishment of a cell expression system for expressing the CRISPR-Cas13a protein and the method for expressing the CRISPR-Cas13a protein in eukaryotic cells are as follows:

1. construction of a vector pDOAL-HFF 1-LshCAs13a expressing LshCAs13a protein

As shown in FIG. 1, the fragment of LshCAs13a coding sequence was amplified from pC001 plasmid by PCR amplification using a pair of C2C2-NcoI-P5 (sequence TATGCATCACCACCATCATCATATGGGGAACCTGTTCGGACACAAG (SEQ ID NO: 1)) and C2C2-NcoI-P3 primer (sequence TCATCGTCGTCCTTGTAGTCCATGGTTACAGGGTATCGTTAGTATTCT (SEQ ID NO: 2)); it was inserted into the pDOAL-HFF 1 plasmid to obtain the recombinant plasmid pDOAL-HFF 1-LshCAs13a.

2. Transformation of plasmids

pDIAL-HFF 1-LshCAs13a was linearized and the linear fragments were transformed into Schizosaccharomyces. 500ng of merozoite cells of the logarithmic growth phase of the linear pDIAL-HFF 1-LshCAs13a fragment were transformed by the lithium acetate/PEG/heat shock method and the LshCAs13a gene was integrated into the LEU1 site of the yeast genome.

3. Protein expression

The recombinant plasmid-transformed schizosaccharomyces was cultured using MM medium to express LshCas13a protein.

Example 2 expression of a gRNA of CRISPR-Cas13a targeting a retrotransposon Tf1 RNA intermediate in eukaryotic cells

Through repeated research, the inventor optimizes the gRNA targeting the Tf1 RNA intermediate and the Gag gene targeting Tf1, wherein the gene sequence is as follows (SEQ ID NO: 12):

5’-atgaaaaactcatcacagaaaagaattcgaatggatggaaatggtggatattgtactcaagatgatatttcagatatccttaagcattttgtaaatcaaaccacccgccatgtggaaacgtatagaaaaggcatggatatggaagagttcatcgttaaattaagaacattttttggtgaacattccgatagatattcaactgaacagtctaaaagactgtacgctatagaacgacttgaatcaagagatcaaaattatgctaataaaatcttttgtcaagattcttctcttacttgggatgaactattaagaagaatggtaaacctagttggatctgatgaagaagaaaggttgactaaaacctttttgaaacttaagaatgataaggacaaggtactattcattaagaaagtactctatgaagataatttaagtgagaaacgagtcagattatatctactatggatgcttccaccctatctgattaaacagagaggtgattcttactgggacatggataaaaatatagacaagatttttaactttgtaccagataaaggtgaaacgataattgaacgctacaccaaacctaggaatcttttaaaaacaaagactggaagcaattggaaaaacaataagtttttaaaggagaacgacactaaagatcgaaaaccaaagaaaacaaatgtttcaaggatcgaatactcatctgaaaattttacaaaatacaagaaaagacgttatgaaa-3’

wherein, starting from the 5' -end, the 306 th and 360 th positions of the nucleic acid are targeted editing sites.

The gRNA was prepared as follows:

1. primers targeting the gRNA of Tf1 RNA intermediate were designed for 3 pairs (2 targets and 1 non-target) and the sequences were as follows (the base at the first 3 of the following sequences was the base matched to the backbone plasmid):

gag gene targeting retrotransposon Tf1 at position 306 (i.e. cleavage between positions 306-307):

Tf1-gRNA-P5(835)：AAC atccaactaggtttaccattcttcttaa(SEQ ID NO:3)；

Tf1-gRNA-P3(835)：GCC ttaagaagaatggtaaacctagttggat(SEQ ID NO:4)；

after the 630 th position of the Gag gene in retrotransposon Tf1 (i.e. cleavage between 630 th and 631 th positions):

Tf1-gRNA-P5(1165)：AAC Tgtcgttctcctttaaaaacttattgtt(SEQ ID NO:5)；

Tf1-gRNA-P3(1165)：GCC aacaataagtttttaaaggagaacgaca(SEQ ID NO:6)；

Tf1-gRNA-P5(Control)：ACC cagactatgcgtcgacaagccaggcatt(SEQ ID NO:7)；

Tf1-gRNA-P3(Control)：GCC aatgcctggcttgtcgacgcatagtctg(SEQ ID NO:8)。

2. construction of plasmid pSK- (LshCAs 13a gRNA) -Tf1

As shown in FIG. 2, primers were synthesized based on the Tf1-gRNA-P5 and Tf1-gRNA-P3 primer sequences provided above, respectively, annealed to form Tf1-gRNA, and inserted into BspQI-digested pSK- (LshCAs 13a gRNA) backbone plasmid to obtain pSK-Tf1-gRNA-28bp-835 plasmid, pSK-Tf1-gRNA-28bp-1165 plasmid, or pSK-Tf1-gRNA-28bp-Control plasmid.

3. Cloning of the expression cassette for gRNA onto the expression plasmid pHL414 of retrotransposon Tf1

As shown in FIG. 3, primers NheI-TYB-P5 and NheI-TYB-P3 were used to amplify the aforementioned pSK-Tf1-gRNA-28bp-835 plasmid, pSK-Tf1-gRNA-28bp-1165 plasmid, or pSK-Tf1-gRNA-28bp-Control plasmid, respectively, to obtain a gRNA expression cassette targeting Tf1 gene; inserted into the pHL414-2 plasmid digested with Nhe, pHL414-Tf1-28bp- (LshCAs 13a gRNA) -835, pHL414-Tf1-28bp- (LshCAs 13a gRNA) -1165, pHL414-Tf1-28bp- (LshCAs 13a gRNA) -Control target plasmids are generated respectively. These target plasmids are abbreviated as pHL414- (LshCas 13a gRNA) -Tf1 plasmids.

NheI-TYB-P5：gggggatcccagctggctagcaattaaccctcactaaagg(SEQ ID NO:9)；

NheI-TYB-P3：tcactatggcgtgctgctagctaatacgactcactatagg(SEQ ID NO:10)。

The previously established pHL414- (LshCAs 13a gRNA) -Tf1 plasmid was transformed into the merozoite-derived strain (containing the integrally expressed LshCAs13a protein) prepared in example 1 described above. 100ng of pHL414- (Lshcas 13a gRNA) -Tf1 plasmid was transformed into merozoite cells at the initial logarithmic growth phase by the method of lithium acetate/PEG/heat shock, leaving the plasmid episomal.

Example 3 test of the Effect of CRISPR-Cas13a interfering retrotransposon Tf1 RNA intermediate in eukaryotic cells such as Schizosaccharomyces

In this example, the effect of interfering with transposition was verified using the previously established model system based on CRISPR-Cas13a intervention to block viral reverse transcription transposition.

The experimental operation steps are as follows:

1. transgenic schizosaccharomyces cerevisiae cells (3 species total) which are established in the previous example and which integrally express LshCAs13a protein and freely express gRNA-Tf1 plasmid are cultivated on MM+thiamine (10. Mu.M) plates and cultivated for 4 days at 32 ℃. Thiamine can inhibit the transposition of the nmt1 promoter to Tf1.

2. Qualitative determination of transposon activity of retrotransposon Tf1

(1) Selecting a monoclonal in an MM+thiamine plate, streaking the monoclonal on a PMG+thiamine (10 mu M) plate, and incubating the monoclonal on the PMG+thiamine plate for 3 days;

(2) Colonies were then transferred to PMG plates (Thianmie was removed to initiate transcription and reverse transcription) and allowed to grow for 4 days;

(3) Colonies were then transferred to plates containing PMG+5-FOA+Uracil+thiamine (free plasmid was removed) and grown for 3 days;

(4) Finally, colonies were transferred to plates containing yes+5-foa+uracil+g418. The transposable activity of Tf1 was monitored by observing whether a derivative yeast strain containing 2 target (targeting Tf1-835, tf1-1165 sites) and 1 non-target (Tf 1-control) episomal plasmids designed by the present inventors was able to continue to grow on G418 plates.

3. Quantitative measurement of transposition frequency of retrotransposon Tf1

(1) The MM+thiamine transformation plates were picked up for monoclonal and transferred to PMG+thiamine (10. Mu.M) plates for incubation for 3 days;

(2) Monoclonal and at initial optical density (OD ₆₀₀ ) Transfer to liquid PMG medium (Thianmie is removed sufficiently to initiate transcription and reverse transcription), allowing the culture to grow for 4 days at 32 ℃,200 rpm;

(3) Inoculating a volume of culture to PMG+5-FOA+Uracil liquid medium to an initial OD ₆₀₀ About 0.1, and culturing for 36 hours.

(4) The culture was then used for 10 ⁷ 、10 ⁶ And 10 ⁵ Each cell/mL was subjected to gradient dilution, and 100. Mu.L of the gradient dilution was inoculated onto a YES+5-FOA or YES+5-FOA+G418 plate, respectively, and incubated at 32℃for 2-3 days.

(5) The number of monoclonal colonies formed on both plates was counted and the frequency of Tf1 retrotransposition was estimated by comparing the colony numbers of plates containing G418+5-FOA with plates containing only 5-FOA.

Transposition frequency (%) = (plate clone number +5-FOA+G418). Times.100/(YES plate clone number +5-FOA). Times.dilution difference (dilution differential)

Colony forming assay results for Tf1-835 and Tf1-1165 as shown in the lower panel of fig. 4, tf1-835 exhibited significantly reduced reverse translocation frequency compared to control (1% ± 0.033%), which had 16% inhibition of Tf1 translocation; the effect of Tf1-1165 on reducing the reverse translocation frequency is relatively more pronounced, with 60% inhibition of Tf1 translocation.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> molecular plant science Excellent innovation center of China academy of sciences

<120> CRISPR-Cas13a based techniques to interfere with blocking viral reverse transcription transposition

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<213> Primer (Primer)

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tatgcatcac caccatcatc atatggggaa cctgttcgga cacaag 46

<210> 2

<211> 48

<212> DNA

<213> Primer (Primer)

<400> 2

tcatcgtcgt ccttgtagtc catggttaca gggtatcgtt agtattct 48

<210> 3

<211> 31

<212> DNA

<213> Primer (Primer)

<400> 3

aacatccaac taggtttacc attcttctta a 31

<210> 4

<211> 31

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<213> Primer (Primer)

<400> 4

gccttaagaa gaatggtaaa cctagttgga t 31

<210> 5

<211> 31

<212> DNA

<213> Primer (Primer)

<400> 5

aactgtcgtt ctcctttaaa aacttattgt t 31

<210> 6

<211> 31

<212> DNA

<213> Primer (Primer)

<400> 6

gccaacaata agtttttaaa ggagaacgac a 31

<210> 7

<211> 31

<212> DNA

<213> Primer (Primer)

<400> 7

acccagacta tgcgtcgaca agccaggcat t 31

<210> 8

<211> 31

<212> DNA

<213> Primer (Primer)

<400> 8

gccaatgcct ggcttgtcga cgcatagtct g 31

<210> 9

<211> 40

<212> DNA

<213> Primer (Primer)

<400> 9

gggggatccc agctggctag caattaaccc tcactaaagg 40

<210> 10

<211> 40

<212> DNA

<213> Primer (Primer)

<400> 10

tcactatggc gtgctgctag ctaatacgac tcactatagg 40

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atgaaaaact catcacagaa aagaattcga atggatggaa atggtggata ttgtactcaa 60

gatgatattt cagatatcct taagcatttt gtaaatcaaa ccacccgcca tgtggaaacg 120

tatagaaaag gcatggatat ggaagagttc atcgttaaat taagaacatt ttttggtgaa 180

cattccgata gatattcaac tgaacagtct aaaagactgt acgctataga acgacttgaa 240

tcaagagatc aaaattatgc taataaaatc ttttgtcaag attcttctct tacttgggat 300

gaactattaa gaagaatggt aaacctagtt ggatctgatg aagaagaaag gttgactaaa 360

acctttttga aacttaagaa tgataaggac aaggtactat tcattaagaa agtactctat 420

gaagataatt taagtgagaa acgagtcaga ttatatctac tatggatgct tccaccctat 480

ctgattaaac agagaggtga ttcttactgg gacatggata aaaatataga caagattttt 540

aactttgtac cagataaagg tgaaacgata attgaacgct acaccaaacc taggaatctt 600

ttaaaaacaa agactggaag caattggaaa aacaataagt ttttaaagga gaacgacact 660

aaagatcgaa aaccaaagaa aacaaatgtt tcaaggatcg aatactcatc tgaaaatttt 720

acaaaataca agaaaagacg ttatgaaa 748

Claims

1. Use of a system for preparing a composition for blocking retrotransposition of a retroorganism in a eukaryotic cell, said blocking retrotransposition of a retroorganism in a eukaryotic cell comprising: preventing the reverse transcribed organism from replicating and jumping in the eukaryotic cell through a reverse transcription step;

the system comprises a Cas13a expression cassette and a crRNA expression cassette, wherein the Cas13a is that the Cas13a is derived from ciliated bacteria #Leptotrichia shahii) LshCas13a; the crRNA targets an essential gene, long terminal repeat, or a region adjacent thereto of the reverse transcribed organism;

in the system, the Cas13a expression cassette is integrated into the genome of the eukaryotic cell for expression, and the crRNA expression cassette is expressed free in the eukaryotic cell.

2. The use according to claim 1, wherein the essential genes comprise genes selected from the group consisting of: structural genes, regulatory genes.

3. The use according to claim 2, wherein the structural or regulatory genes comprise: gag, PR, RT, IN, env, tat, rev, vpu, nef, vpr or Vpx.

4. A use according to any one of claims 1 to 3, wherein the reverse transcribed organism comprises: retrovirus, a mimic of retrovirus.

5. The use according to claim 4, wherein said mimetic of a retrovirus is retrotransposon Tf1.

6. The use of claim 5, wherein said retrotransposon Tf1 comprises: a retrotransposition element for detecting a screening gene for retrotransposition events.

7. The use according to claim 6, wherein the reverse seat element comprises an operatively linked gene selected from the group consisting of: long terminal repeats, gag, PR, RT, IN; the long terminal repeat includes a 5'LTR and a 3' LTR.

8. The use according to claim 6, wherein the screening gene is a resistance screening gene, a marker gene or a reporter gene.

9. The use according to claim 8, wherein the resistance gene is Neo.

10. The use of claim 6, wherein said retroviral mimetic further comprises a promoter operably linked to said retrotransposon that drives transposition of said retrotransposon.

11. The use according to claim 10, wherein the promoter is a regulatable promoter which isnmt1A promoter that is inhibited by thiamine.

12. The use of claim 1, wherein the crRNA expression cassette comprises the following elements operably linked: direct repeat sequence, gRNA.

13. The use of claim 12, wherein the crRNA expression cassette comprises the following elements operably linked: promoters, direct repeats, gRNA and ribozymes.

14. The use according to claim 13, wherein the promoter isrrk1A promoter.

15. The use according to claim 13, wherein the nucleotide sequence of the direct repeat is shown in SEQ ID No. 11.

16. The use of claim 3, wherein the structural or regulatory gene comprises Gag and the crRNA targets position 306 or 360 of the Gag gene.

17. The use of claim 13, wherein the gRNA is selected from the group consisting of: nucleotide sequences at positions 4-31 in the sequence shown in SEQ ID NO. 3 or 5, or their reverse complements.

18. The use according to claim 1, wherein the eukaryotic cellWhen the cell is yeast, the Cas13a expression cassette is integrated into yeast chromosome IILEU1A site.

19. A method of blocking retrotransposition of a retroorganism in a eukaryotic cell, said blocking retrotransposition of a retroorganism in a eukaryotic cell comprising: preventing the reverse transcribed organism from replicating and jumping in the eukaryotic cell through a reverse transcription step; the method comprises the following steps:

(1) Providing a system; the system comprises a Cas13a expression cassette and a crRNA expression cassette, wherein the Cas13a is derived from ciliates #Leptotrichia shahii) LshCas13a; the crRNA targets an essential gene, long terminal repeat, or a region adjacent thereto of the reverse transcribed organism; in the system, the Cas13a expression cassette is integrated into the genome of the eukaryotic cell for expression, and the crRNA expression cassette is expressed freely in the eukaryotic cell;

(2) Introducing the system of (1) into a eukaryotic cell, whereby the eukaryotic cell has the ability to block retrotransposition of a retroactive organism.

20. The method of claim 19, wherein the eukaryotic cell is a yeast cell, a plant cell, or an animal cell.

21. The method of claim 20, wherein the yeast cells comprise schizosaccharomyces cells.