CN119899837A

CN119899837A - A DNA lead editing system, recombinant vector, kit and method for inverted target DNA sequence

Info

Publication number: CN119899837A
Application number: CN202510016521.4A
Authority: CN
Inventors: 殷昊; 张傲; 张楹; 孙祥堃
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2025-01-06
Filing date: 2025-01-06
Publication date: 2025-04-29

Abstract

The present invention discloses a DNA lead editing system, a recombinant vector, a kit and a method for an inverted target DNA sequence, and relates to the field of gene editing technology. The DNA lead editing system includes a Cas protein and a reverse transcriptase, pegRNA-A and pegRNA-B; pegRNA-A includes a first guide RNA, a first primer binding site and a first reverse transcription template, and pegRNA-B includes a second guide RNA, a second primer binding site and a second reverse transcription template; the first reverse transcription template includes a first paired fragment, and the second reverse transcription template includes a second paired fragment; the first paired fragment is complementary to the sequence of the second paired fragment; pegRNA-A and pegRNA-B respectively guide the Cas protein to cut the target fragment at the restriction sites on both sides of the same strand of double-stranded DNA. The present invention provides four sets of DNA lead editing systems based on the same technical concept. These four sets of DNA lead editing systems all generate 3' single-stranded DNA at both ends of the target DNA sequence through specially designed pegRNA, and realize the inverted editing of the target DNA through the complementary action of the paired fragments.

Description

DNA leader editing system, recombinant vector, kit and method for inverted target DNA sequence

Technical Field

The invention relates to the technical field of gene editing, in particular to a DNA leader editing system, a recombinant vector, a kit and a method for inversed target DNA sequences.

Background

CRISPR-based genome editing tools drastically change our unprecedented ability to manipulate genomic sequences with precision. CRISPR-Cas nucleases induce site-specific Double Strand Breaks (DSBs) through repair mechanisms such as non-homologous end joining (NHEJ) or Homology Directed Repair (HDR) to achieve targeted genomic modification. These pathways facilitate small insertions/deletions (indels) or precise modification using donor templates.

In recent years, catalytically impaired Cas nucleases have been designed to form a Base Editor (BE) in conjunction with deaminase or to develop a lead editor (PE) in conjunction with Reverse Transcriptase (RT). The base editor allows for efficient base substitution at a particular genomic site without relying on HDR or donor DNA. On the other hand, the leader editor uses leader editing guide RNAs (pegrnas) to direct precise sequence modification through the RT templates encoded within pegRNA. By combining PE with integrase, the precise DNA modification can range from a few base pairs (bp) to about 40 kb, which covers the average length of a single gene.

A large part of pathogenic mutations originate from structural variations in the genome. Although these variations can be induced by using Cas9 nuclease in combination with a pair of guide RNAs (grnas), the main editing result is typically small insertions/deletions, with structural variations being relatively minor byproducts. Despite the success of precise editing at a single genetic locus, the development of tools that can accurately and efficiently design large-scale genomic structural variations of mammalian cells remains a critical unmet need.

In contrast to deletions and duplications, inversions do not generally alter the genome copy number, but can greatly affect gene expression and genome integrity. These rearrangements are critical for understanding human evolution and are associated with various genetic diseases such as hemophilia a, mucopolysaccharidosis type II (hunter syndrome) and emery-Lei Fusi muscular dystrophy, which are usually caused by inversions between inverted homologous sequences. Furthermore, inversion can lead to the formation of oncogenes, such as ALK-EML4 fusion associated with lung cancer, emphasizing their potential impact on gene function. Despite their association with genetic diseases and various cancers, there is still a lack of efficient and convenient genetic tools to mimic chromosomal inversion in cells and animals. Although binding of Cas9 nuclease to the gRNA pair can induce inversion, these events are not common and are typically masked by insertions/deletions. More recent methods, such as combining PE with an integrase and using two pairs pegRNA to install recombination sites, have been shown to be able to invert the 40 kb fragment. However, these methods are still inefficient for inversion in mammalian cells and require time consuming continuous transfection to install recombination sites prior to inversion. Given the critical role of inversion in genomic structure and disease, there is a strong need for a more efficient and programmable tool to manipulate and study these structural variations.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a DNA leader editing system, a recombinant vector, a kit and a method for inversing a target DNA sequence, which can realize inversed editing of the target DNA sequence more accurately and more efficiently by using leader editing guide RNA (pegRNA). Specifically, the method is realized by the following technology.

In a first aspect of the invention, there is provided a first DNA-guided editing system for inverting a target DNA sequence, the DNA-guided editing system comprising a Cas protein and a reverse transcriptase, pegRNA-A and pegRNA-B;

The pegRNA-A comprises a first guide RNA, a first primer binding site, and a first reverse transcription template, and the pegRNA-B comprises a second guide RNA, a second primer binding site, and a second reverse transcription template;

The first reverse transcription template comprises a first pairing fragment, and the second reverse transcription template comprises a second pairing fragment, wherein the sequences of the first pairing fragment and the second pairing fragment are complementary;

the pegRNA-A and the pegRNA-B each direct the Cas protein to cleave the fragment of interest at a cleavage site flanking the same strand of double-stranded DNA.

Further, the sequence length of the first reverse transcription template and the second reverse transcription template is 0 to 2,000 nucleotides, preferably 15 to 500 nucleotides.

Further, the first and second mating fragments have a sequence length of 0 to 1,000 nucleotides, preferably 3 to 200 nucleotides, and more preferably 30 to 100 nucleotides.

Further, the first reverse transcription template further comprises 1 first non-complementary template sequence, the second reverse transcription template further comprises 1 second non-complementary template sequence, and the first non-complementary template sequence and the second non-complementary template sequence are not complementary to each other.

Still further, the first non-complementary template sequence, the second non-complementary template sequence, is 1 to 2,000 nucleotides in length, preferably 1 to 1,000 nucleotides in length, more preferably 1 to 500 nucleotides in length.

Further, the cleavage sites located on both sides of the same strand of the double-stranded DNA are spaced apart by 500 to 100,000,000 base pairs, preferably 10,000 to 30,000,000 base pairs.

Further, the pegRNA-A and/or pegRNA-B also include a tail sequence that forms a hairpin or loop with itself, or that includes a poly (A), poly (U), or poly (C) sequence, or that includes an RNA binding domain.

The DNA leader editing system provided by the invention introduces two complementary 3' flaps (PRIME EDITING-based Inversion WITH ENHANCED performance version 1, PIEv 1) on the same strand of double-stranded DNA by using Cas protein, reverse transcriptase RT and two pegRNA (A and B). When the two complementary single stranded DNA sequences are joined together, inversion occurs under the repair of the DNA (FIG. 1). This method is referred to herein as PIEv method/design.

In a second aspect of the present invention, there is provided a second DNA leader editing system for inverting a target DNA sequence, characterized in that the DNA leader editing system comprises Cas protein and reverse transcriptase, pegRNA-A, pegRNA-B, pegRNA-C, pegRNA-D;

The pegRNA-A comprises a first guide RNA and a first reverse transcription template, the pegRNA-B comprises a second guide RNA and a second reverse transcription template, the pegRNA-C comprises a third guide RNA, a third primer binding site and a third reverse transcription template, and the pegRNA-D comprises a fourth guide RNA, a fourth primer binding site and a fourth reverse transcription template;

the first reverse transcription template comprises a first pairing fragment, the second reverse transcription template comprises a second pairing fragment, the third reverse transcription template comprises a third pairing fragment and the fourth reverse transcription template comprises a fourth pairing fragment;

The pegRNA-A and the pegRNA-B respectively guide the Cas protein to cut the target fragment at the enzyme cutting sites at two sides of one strand of the double-stranded DNA, and the pegRNA-C and the pegRNA-D respectively guide the Cas protein to cut the target fragment at the enzyme cutting sites at two sides of the other strand of the double-stranded DNA;

The pegRNA-A motif proximal to the protospacer sequence of the cleavage site that directs cleavage of the Cas protein is disposed proximal to (PAM-in form) or distal to (PAM-out form) the Spacer sequence relative to the motif proximal to the pegRNA-C motif proximal to the protospacer sequence of the cleavage site that directs cleavage of the Cas protein.

In the present invention, the PAM-out format is shown in fig. 7. In FIG. 7, the primordial Spacer adjacent motif (PAM) of the cleavage site of the corresponding Cas protein of pegRNA-A is to the right of the Spacer sequence (ProtospacerA), the primordial Spacer adjacent motif (PAM) of the cleavage site of the corresponding Cas protein of pegRNA-C is to the left of the Spacer sequence (ProtospacerB), and the two PAMs of pegRNA-A and pegRNA-C are located far apart from each other relative to the two Spacer sequences. Similarly, the two PAMs pegRNA-B and pegRNA-D are also located far apart from each other relative to the two Spacer sequences.

In the present invention, the PAM-in form is shown in FIG. 9. In FIG. 9, the primordial Spacer adjacent motif (PAM) of the cleavage site of the corresponding Cas protein of pegRNA-A is located to the right of the Spacer sequence (ProtospacerA), the primordial Spacer adjacent motif (PAM) of the cleavage site of the corresponding Cas protein of pegRNA-C is located to the left of the Spacer sequence (ProtospacerB), and the two PAMs of pegRNA-A and pegRNA-C are located close to each other relative to the two Spacer sequences. Similarly, the two PAMs pegRNA-B and pegRNA-D are also disposed close to each other relative to the two Spacer sequences.

Further the sequence length between A and C is from 2 to 10,000, preferably 500 to 2,000 nucleotides.

Further the sequence length between B and D is from 2 to 10,000, preferably from 500 to 2,000 nucleotides.

Further, the cleavage sites located on both sides of the same strand of the double-stranded DNA are 500 to 100,000,000 base pairs apart;

still further, from 10,000 to 30,000,000 base pairs apart.

To develop a precise inversion editing tool, we tried to introduce a second pair of complementary 3' single stranded DNA on the other strand of double stranded DNA in addition to ensure the precision of the other end interface. For two adjacent/nearby pegRNA (pegRNA-A and pegRNA-C, and pegRNA-B and pegRNA-D in fig. 7 and 8), there are two forms of PAM-out and PAM-in. Herein, "adjacent" refers to two pegRNA located on two DNA strands and adjacent to each other. For example, pegRNA-A and pegRNA-C are defined as two "adjacent/contiguous" sets of pegRNAs, and pegRNA-B and pegRNA-D are defined as two "adjacent/contiguous" sets of pegRNA ". Thus, the above second DNA leader editing system provided by the present invention is practically divided into two more specific sets of DNA leader editing systems, designated PIEv method/design and PIEv method/design in the present invention, respectively, for both the PAM-out and PAM-in forms.

For the PAM-out format, PIEv method/design targets four sites on the genomic double-stranded DNA, two complementary pairs of 3' single-stranded DNA were synthesized by reverse transcriptase RT. Inversion occurs under the action of DNA repair when the two complementary pairs of 3' single stranded DNA are bound together, respectively. After the inversion editing is completed, four original targets still exist.

For the case of PAM-in format, the PIEv method/design also targets four sites on the genomic double stranded DNA, two complementary pairs of 3' single stranded DNA were synthesized by RT. When the two complementary pairs of 3' -single-stranded DNA are respectively combined together, inversion occurs under the action of DNA repair, and simultaneously, the sequence between two nick at the two ends of the same side is deleted, so that the occurrence of secondary editing is prevented.

Compared with PIEv method/design, PIEv method/design and PIEv method/design, the inversion editing can be realized more accurately.

In a third aspect of the invention, there is also provided a DNA leader editing system for inverting a target DNA sequence, the DNA leader editing system comprising Cas protein and reverse transcriptase, pegRNA-E and pegRNA-F;

the pegRNA-E comprises a fifth guide RNA, a fifth primer binding site and a fifth reverse transcription template, and the pegRNA-F comprises a sixth guide RNA, a sixth primer binding site and a fifth reverse transcription template, wherein the fifth guide RNA and the sixth guide RNA respectively target a target fragment;

The fifth pairing fragment is complementary with the sequence of the gene fragment, which is adjacent to the motif near one side of the 3 'end and is next to the cleavage site of the Cas protein, of the sixth guide RNA, and the sixth pairing fragment is complementary with the sequence of the gene fragment, which is adjacent to the motif near one side of the 3' end and is next to the cleavage site of the Cas protein, of the fifth guide RNA;

The pegRNA-E and the pegRNA-F respectively guide the Cas protein to cut the enzyme cutting site of the target fragment on two sides of two opposite strands of double-stranded DNA, and the adjacent motif of the original spacing sequence of the enzyme cutting site of the Cas protein by the pegRNA-E are far away relative to the corresponding Spacer sequence.

Further, the cleavage sites located on both sides of the two opposite strands of the double-stranded DNA are separated by 500 to 50,000,000 base pairs, preferably 1,000 to 500,000 base pairs.

It can be appreciated that in the above-mentioned several DNA leader editing systems provided by the present invention, the first guide RNA, the second guide RNA, the third guide RNA, the fourth guide RNA, the fifth guide RNA and the sixth guide RNA respectively target the respective corresponding target fragments. The terms "first", "second", "third", "fourth", "fifth" and "sixth" are merely used for distinguishing, and do not create any special technical limitations. The guide RNA can be a specific sgRNA or crRNA for different CRISPR/Cas gene editing systems.

For example, in a CRISPR/Cas9 gene editing system, the guide RNA is sgRNA, and in a CRISPR/Cas12 or CRISPR/Cas13 gene editing system, the guide RNA is crRNA.

In a fourth aspect of the invention, there is provided a substance, any one of:

(1) A nucleic acid composition comprising a gene encoding any one of the DNA leader editing systems provided above in accordance with the present invention;

(2) A recombinant vector comprising the coding gene of any one of the DNA leader editing systems provided above;

(3) A recombinant microorganism which encodes the gene encoding any one of the DNA leader editing systems provided above;

(4) A kit comprising any one of the DNA lead editing systems provided above of the invention, or comprising the recombinant vector.

Further, when the recombinant vector includes the coding gene of the DNA leader editing system provided in the second aspect of the present invention, the pegRNA-A and pegRNA-D are recombined on the same recombinant vector, and the pegRNA-B and pegRNA-C are recombined on the same recombinant vector.

PegRNA is typically cloned separately on different vectors when applied using PIEv method/design, PIEv2 method/design, or PIEv3 method/design. When such a vector construction method is employed, there may be a phenomenon that plasmid distribution is not uniform after transfection of cells. Especially when PIEv methods/designs are used, this may lead to PIEv1 editing results, resulting in inaccurate inversions.

To reduce the occurrence of the above problems, for the PIEv method/design, the present invention clones two pegRNA (pegRNA-A and pegRNA-D) distal and targeting different DNA strands on the same vector, and the other two pegRNA (pegRNA-B and pegRNA-C) on the same vector. These two vectors do not undergo any editing when only one vector is present in the cell, and only two vectors are simultaneously present in the cell, the editing of PIEv.

In a fifth aspect of the invention, there is provided the use of a DNA leader editing system as defined in any one of the preceding claims for inverting a target DNA sequence.

In a sixth aspect of the present invention, there is provided a method of inverting a target DNA sequence comprising the steps of:

Adding the target DNA sequence and any one of the DNA leader editing systems provided in the first aspect of the invention into a reaction system, wherein the reverse transcriptase respectively carries out reverse transcription by using the first reverse transcription template and the second reverse transcription template to generate 2 single-stranded DNA sequences, and the 2 single-stranded DNA sequences are complementarily combined to form a double-stranded region;

Or comprises the following steps:

Adding the target DNA sequence and the DNA leader editing system provided by the second aspect of the invention into a reaction system, wherein the reverse transcriptase respectively reverse transcribes the first reverse transcription template, the second reverse transcription template, the third reverse transcription template and the fourth reverse transcription template to generate 4 single-stranded DNA sequences, and the 4 single-stranded DNA sequences are complementarily combined to form 2 double-stranded regions;

When the two PAMs (pro-Spacer adjacent motif of cleavage site of Cas protein cleavage) of pegRNA-A and pegRNA-C are in a form distant from each other relative to the two Spacer sequences (pegRNA-B and pegRNA-D homology), i.e. PAM-out form, inverting the target DNA sequence based on DNA repair mechanism, inserting the complementary DNA sequence of the first mating fragment near the interface of the first mating fragment and increasing in situ the sequence copy between a and C;

When the two PAMs (pro-Spacer adjacent motif of cleavage site of Cas protein cleavage) of pegRNA-A and pegRNA-C are in the form of PAM-in relative to the two Spacer sequences, in the form of reciprocal approach (pegRNA-B and pegRNA-D homology), the target DNA sequence is inverted based on DNA repair mechanism, deleting the sequence copy between a and C and inserting the complementary DNA sequence of the first mating segment near the interface of the first mating segment;

Or comprises the following steps:

Adding the target DNA sequence and the DNA leader editing system provided in the third aspect into a reaction system, carrying out reverse transcription by the reverse transcriptase through the fifth reverse transcription template and the sixth reverse transcription template to generate 2 single-stranded DNA sequences, respectively combining the 2 single-stranded DNA sequences with corresponding complementary genome regions to form double-stranded regions, and reversing the target DNA sequence based on a DNA repair action mechanism.

Further, in the above method of inverting the target DNA sequence, the Cas protein is a Cas9 protein containing an inactivated HNH domain, or a Cas9 protein containing an inactivated RuvC domain.

Still further, in the above method of inverting a target DNA sequence, the Cas protein is SpCas9、FnCas9、St1Cas9、St3Cas9、NmCas9、SaCas9、VQR SpCas9、EQR SpCas9、VRER SpCas9、SpCas9-NG、xSpCas9、RHA FnCas9、KKH SaCas9、NmeCas9、StCas9、CjCas9 or AtCas.

Further, in the above method of inverting the target DNA sequence, the Cas protein is a Cas12 protein.

Further, in the method for inverting the target DNA sequence, the Cas12 protein is Cas12a, cas12b, cas12f or Cas12i.

In a specific alternative, in the above method of inverting the target DNA sequence, the Cas12 protein is one or more of AsCpf1、LbCpf1、FnCpf1、SsCpf1、PcCpf1、BpCpf1、CmtCpf1、LiCpf1、PmCpf1、Pb3310Cpf1、Pb4417Cpf1、BsCpf1、EeCpf1、BhCas12b、AkCas12b、EbCas12b、LsCas12b.

Alternatively, the target DNA sequence is located within a cell.

Alternatively, the cell is a dividing cell or a non-dividing cell.

Alternatively, the target DNA sequence is a telomere or fragment thereof.

Alternatively, the above method of inverting the target DNA sequence is performed in vitro or in vivo.

Optionally, each pegRNA includes a first or second guide RNA, a first or second primer binding site, a first or second reverse transcription template, arranged in a "5 'to 3'" or "3 'to 5'" direction.

Alternatively, the reverse transcriptase is an M-MLV reverse transcriptase or a reverse transcriptase capable of functioning under physiological conditions.

Alternatively, the Cas protein and reverse transcriptase are provided indirectly as nucleic acid fragments encoding the respective proteins, or directly as proteins.

Alternatively, each pegRNA is provided indirectly as recombinant DNA encoding the pegRNA, or directly as an RNA molecule.

Compared with the prior art, the invention has the advantage that the invention provides four sets of DNA pilot editing systems based on the same technical conception. The four sets of DNA leader editing systems all generate complementary pairing fragments at two ends of the same strand of a target DNA sequence through specially designed leader editing guide RNA (pegRNA), and realize inversion editing of the target DNA through the complementary action of the pairing fragments.

The four sets of DNA lead editing systems provided by the invention can aim at any target DNA sequence needing inversion editing, and are not limited to a specific species or class of species. In practical application, the four sets of DNA pilot editing systems can obtain good gene inversion editing effects by adopting the technical conception of the invention for corresponding species.

Drawings

FIG. 1 is a schematic diagram showing the process of achieving inversion of a target DNA by the method PIEv of example 1. The PE2-pegRNA complex targets two sites on the same strand of genomic double-stranded DNA. Two complementary 3' single stranded DNA were synthesized by RT using RTT that does not have homology to the genomic sequence. Inversion (yellow part) occurs under DNA repair when two complementary 3' single stranded DNA anneal together.

FIG. 2 is a TAE gel electrophoresis chart of the result of detecting inversion in example 1, confirming inversion of 10 kb, 100 kb, and 1 Mb on chromosome 6.

FIG. 3 shows the results of one-generation sequencing (Sanger sequencing) of the inverted junction sequences of example 1, with the sequences on the left and right of the 1Mb inverted junction on chromosome 6, with precise left and random insertions and deletions on the right.

Fig. 4 and 5 are verification results of integrity of inversion occurrence in example 1. Sanger sequencing of FIG. 4 verifies the wild-type genome in HEK293T cells and FIG. 5 verifies that the VEGFA site occurs 836 bp inversion.

FIG. 6 is the inversion editing efficiency of PIEv1 of example 1 in HEK293T and N2a cells.

FIG. 7 is a schematic diagram showing the process of achieving inversion of a target DNA by the method PIEv of example 2. The PE2-pegRNA complex targets four sites on genomic double stranded DNA. Two complementary pairs of 3' single stranded DNA were synthesized by RT. When the two complementary pairs of 3' single stranded DNA are respectively bound together, inversion (yellow part) occurs under the action of DNA repair. After the inversion editing is completed, four target sites remain.

FIG. 8 is a schematic diagram of the second editing after PIEv.sup.2 inversion editing in example 2, and Amplification Editing (AE) on the genomic DNA after PIEv editing. When the inversion editing is completed, four original target sites remain, and then two complementary pairs of 3' single-stranded DNA are resynthesized on different strands of double-stranded DNA, inducing AE.

FIG. 9 is a schematic diagram showing the process of achieving inversion of a target DNA by the method PIEv of example 2. The PE2-pegRNA complex targets four sites on genomic double stranded DNA. Two complementary pairs of 3' single stranded DNA were synthesized by RT. When the two complementary pairs of 3' single stranded DNA are respectively bound together, inversion occurs under the action of DNA repair while deleting part of the fragments at both ends (grey parts).

FIG. 10 is a TAE gel electrophoresis chart of the inversion result detected by the method PIEv in example 2, and the inversion of 11 kb and 1 Mb on chromosome 6 was determined.

FIG. 11 shows the results of one-generation sequencing (Sanger sequencing) of the sequence at the interface after inversion by the method PIEv in example 2, and the amplification edits after 10kb inversion on chromosome 6 were determined.

FIG. 12 is a TAE gel electrophoresis chart of the result of inversion detection by PIEv in example 2, which shows that 10 kb, 100kb and 1 Mb were inverted on chromosome 6.

FIG. 13 shows the results of one-generation sequencing (Sanger sequencing) of the inverted sequence at the junction using the method PIEv in example 2, and the determination of the inverted sequence at the left and right junctions of chromosome 6, 1 Mb, was accurate on both the left and right sides.

FIG. 14 shows the product purity of the two-terminal interface sequences after inversion of the 10 kb to 30 Mb region on chromosome 6 in HEK293T (FIG. 14 a) and K562 (FIG. 14 b) cells and the efficiency of inversion of the 10 kb to 30 Mb region on chromosome 6 in HEK293T (FIG. 14 c) and K562 (FIG. 14 d) cells, as quantified by ddPCR, in example 2.

Fig. 15 and 16 are schematic views showing the reduction of PIEv b in non-precision editing in example 3.

FIG. 17 shows the results of the inversion editing efficiency verification of PIEv b in different cell lines in example 3.

FIG. 18 is a target inversion at the EGFP site for a functional cell line in example 3. FIG. 18a is a schematic diagram of GFP-function recovering cell lines. FIG. 18b is an edit efficiency of the inversion of 957 bp in the EGFP reverse-inactivation region, quantified by flow cytometry.

FIG. 19 is an in-arm inversion event on chromosome 6 determined by fluorescence in situ hybridization in example 3. Fluorescent In Situ Hybridization (FISH) mapping of the inversion of 40 Mb on chromosome 6. Column 1 from the left represents the centromere of chromosome 6 (green). In column 2, the red fluorescent probe labeled the genomic sequence of about 198 kb surrounding the ESR1 gene. Columns 1 and 2 are combined in column 3, with the core shown in blue. Column 4 is an enlarged view of column 3. Edit-1 and Edit-2 are different HEK293T monoclonal cells with the 40 Mb inversion

FIG. 20 shows the effect of the DNA guide editing system of example 4 on the inversion editing of hemophilia A and Acute Myelogenous Leukemia (AML) sites in HEK293T cells.

FIG. 21 is a schematic representation of a human chromosome possessing a terminal centromere structure created by IE technology in example 4.

FIG. 22 shows the use of FISH to detect the 100 Mb inversion on Chr 2 in example 4. Column 1 (from the left) shows the Chr 2 centromere satellite DNA region marked with green. Column 2 combines column 1 and DAPI staining, with nuclei shown in blue. Column 3 provides an enlarged view of column 2. The edition-1 and edition-2 are different single cell clones from IE treated samples for 100 Mb inversions on Chr 2 in HEK293T cells.

FIG. 23 shows the detection of a 30 Mb inversion on Chr 20 using FISH. Column 1 (from the left) shows the genomic sequence of 730. 730 kb near the green fluorescent probe marker MARPE gene. Column 2 shows the genomic sequence of 379 kb near the red fluorescent probe marker YWHAB gene. Columns 1 and 2 are combined in column 3 and the nuclei stained blue. Column 4 is an enlarged view of column 3, column 5 shows only an enlarged view of DAPI staining. Edit-1 and Edit-2 represent different single cell clones from IE-treated samples, with 30 Mb inversions on Chr 20 in HEK293T cells.

FIG. 24 is a schematic diagram showing the implementation of the target DNA inversion by PIES-PAM-out method in example 5.

FIG. 25 is a graph showing the efficiency of the inversion of 100 kb and 1 Mb on chromosome 6 and the inversion of 10 Mb on chromosome 2 in HEK293T (a) and K562 (b) cells in example 5.

FIG. 26 is a purity statistic of both end-to-end interfaces after inversion of the 100 kb and 1Mb chromosome 6 in HEK293T cells, as described in example 5.

Detailed Description

The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are only some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.

It should be emphasized that the following specific embodiments only exemplify the four sets of DNA pilot editing systems provided by the present invention to implement gene inversion editing, and the corresponding inversion editing effect can not be obtained only for HEK293T cells, but also not only by adopting the following specific DNA pilot editing systems.

The four sets of DNA leader editing systems of the present invention are not limited to Cas protein, reverse transcriptase or pegRNA sequences specific to the following embodiments, nor are the target DNA sequences targeted to be limited to a particular species or class of species. The target DNA sequence to be inverted edited is not limited to a specific species or class of species. In practical application, the four sets of DNA pilot editing systems can obtain good gene inversion editing effects by adopting the technical conception of the invention for corresponding species.

Example 1 design and feasibility verification of PIEv1

In order to realize accurate and efficient inversion editing of the target DNA, the embodiment uses a compound of PE2 and two pegRNA to perform inversion editing of the target DNA.

1. PIEv1 design of 1

As shown in fig. 1. Two complementary 3 'single stranded DNA (Inversion editing version, 3' flap) was introduced by Reverse Transcriptase (RT) using a Reverse Transcription Template (RTT) that did not have homology to the genomic sequence, targeting two sites on the same strand of genomic double stranded DNA using a complex consisting of PE2 (nCas-RT) and two pegRNA (pegRNA A and pegRNA B).

When the two complementary 3' flaps are annealed together, inversion occurs under the repair of the DNA (yellow part in fig. 1). This target DNA inversion method was designated PIEv as design.

2. PIEv1 feasibility verification of 1

To verify the feasibility of PIEv1, this example attempted 10kb, 100kb and 1Mb inversions on chromosome 6 in HEK293T cells. The specific method comprises the following steps:

The sequence of the PE2 plasmid is shown as SEQ ID NO. 1.

The nucleotide sequence of pegRNA plasmid is shown in SEQ ID NO.2, wherein 3253-3500 is promoter sequence, 3501-3520 is spacer sequence, 3521-3596 is sgRNA skeleton sequence, 3597-3639 is RTT and PBS sequence, 3640-3684 is linker and evopreQ motif sequence, wherein n is merogenesis base, namely representing any 1 base in A, T, C, G.

For different inversion events, the spacer sequence at bits 3501-3520 and the RTT and PBS sequences at bits 3597-3639 are different. The details are shown in table 1 below.

TABLE 1

The pegRNA plasmid and PE2 plasmid were co-transfected into HEK293T cells and the genomes were harvested after 3 days of culture. The occurrence of inversion event is detected by designing two pairs of primers at the interfaces of the two ends of the inversion region, respectively. Only the inverted sequence can amplify the band of interest. The relevant primers are shown in Table 2 below.

TABLE 2

As shown in the TAE gel electrophoresis chart of FIG. 2, the bands could not be amplified by using the F1/F2 or R1/R2 primer pair on the genome (WT group) without any plasmid transfected, and the bands of interest could be amplified by using the F1/F2 or R1/R2 primer pair in the genome in which inversion occurred after editing, indicating that 10 kb, 100 kb and 1 Mb inversion was achieved on chromosome 6 of HEK293T cells.

As shown in FIG. 3, by first-generation sequencing (Sanger sequencing) the inverted sequence at the interface, it was found that the insertion of the flap sequence was carried out at the left interface, ensuring the accuracy under the action of the flap complement, while the obvious mutation (Indel) was present at the right interface, in particular with random insertions and deletions. The PIEv method has some non-precise editing.

To verify the integrity of the inversion, we performed 836 bp inversions of the VEGFA gene on chromosome 6. The genome was harvested after transfection of 293T cells, and the amplified inversion bands were used to construct T/a clones, confirming the 836 bp inversion of the VEGFA site (fig. 4), and the integrity of inversion was confirmed by first generation sequencing (Sanger sequencing) (fig. 5). The nucleotide sequence of pegRNA plasmid used is also shown in SEQ ID NO. 2. For different inversion events, the spacer sequence at bits 3501-3520 and the RTT and PBS sequences at bits 3597-3639 are different. The details are shown in Table 3 below.

TABLE 3 Table 3

The amplification primer VEGFA-inv 836 bp-F is 5'-ctctttagccagagccgggg-3', and is shown in SEQ ID NO. 23.

The amplification primer VEGFA-inv 836 bp-R is 5'-ggtggagggggtcggggct-3', and is shown as SEQ ID NO. 24.

3. PIEv1 cell line validation of 1

To determine the efficiency of PIEv1 editing in cell lines, we inverted different regions on different chromosomes in human HEK293T cells and mouse N2a cells, respectively. pegRNA used in human HEK293T cells is also shown in SEQ ID NO. 2. For different inversion events, the spacer sequence at bits 3501-3520 and the RTT and PBS sequences at bits 3597-3639 are different. In mouse N2a cells, the spacer sequence and RTT and PBS sequences of the different inversion events are shown in table 4 below.

TABLE 4 Table 4

The pegRNA plasmid and PE2 plasmid were co-transfected cells and the genomes were harvested 3 days later and ddPCR was used to identify the inversion efficiency at the interface. The results show that efficiencies in HEK293T and N2a cells are 32.8-38.4% and 0.4-10.1%, respectively (a and b of fig. 6), demonstrating that PIEv1 can achieve efficient inversion editing, but is less efficient (1 Mb or more) on chromosome level editing.

Example 2 design and feasibility verification of PIEv2 (PAM-in) and PIEv (PAM-out)

In order to achieve more accurate inversion editing of the target DNA, we tried to introduce a second pair of complementary 3' flaps on the other strand of the double-stranded DNA to ensure the accuracy of the other end interface. That is, this example also selects 2 pegRNA (pegRNA C and pegRNA D) in addition to the 2 pegRNA (pegRNA A and pegRNA B) provided in example 1.

As shown in fig. 7 and 9, there are two forms of PAM-in (as shown in fig. 9) and PAM-out (as shown in fig. 7) for two pegRNA on the same side.

In FIG. 7, the primordial Spacer adjacent motif (PAM) of the cleavage site of the corresponding Cas protein of pegRNA-A is to the right of the Spacer sequence (ProtospacerA), the primordial Spacer adjacent motif (PAM) of the cleavage site of the corresponding Cas protein of pegRNA-C is to the left of the Spacer sequence (ProtospacerB), and the two PAMs of pegRNA-A and pegRNA-C are in a form distant from each other relative to the two Spacer sequences. Similarly, the two PAMs pegRNA-B and pegRNA-D are also in a form that is remote from each other relative to the two Spacer sequences.

In FIG. 9, the primordial Spacer adjacent motif (PAM) of the cleavage site of the corresponding Cas protein of pegRNA-A is located to the right of the Spacer sequence (ProtospacerA), the primordial Spacer adjacent motif (PAM) of the cleavage site of the corresponding Cas protein of pegRNA-C is located to the left of the Spacer sequence (ProtospacerB), and the two PAMs of pegRNA-A and pegRNA-C are in close proximity to each other relative to the two Spacer sequences. Similarly, the two PAMs pegRNA-B and pegRNA-D are also in close proximity to each other relative to the two Spacer sequences.

The following two types of target DNA inversion methods are respectively designed in the form of PAM-in and PAM-out.

1. Target DNA inversion method with PAM-out form of two pegRNA on the same side

(1) PIEv2 design

As shown in FIG. 7, the method is designed in such a way that two pegRNA on the same side are in the form of PAM-out, a complex consisting of nCas-RT and 4 pegRNA is targeted to four sites on the genome double-stranded DNA, and two pairs (four) of complementary 3' flaps are introduced by Reverse Transcriptase (RT) using Reverse Transcription Templates (RTT) which do not have homology with the genome sequence.

When the two complementary pairs of 3' flaps are annealed together, inversion is completed under the repair mechanism (yellow part in FIG. 7). The accuracy of the interfaces at the two ends is ensured by two pairs of complementary 3' flaps, and the design of the target DNA inversion method is PIEv.

As shown in FIG. 8, amplification Editing (AE) occurred on the genomic DNA edited by PIEv 2. When the inversion editing is completed, four original targets (Protospacer-A, B, C and D) remain, and the A and D positions are interchanged. Two complementary pairs of 3' single-stranded DNA were then resynthesized on both strands of the double-stranded DNA, and the inverted double-end interface was subjected to AE.

(2) PIEv2 feasibility verification of 2

Using the PIEv2 design, this example inverted the 10kb and 1Mb region on chromosome 6 in 293T cells. The specific method comprises the following steps:

The nucleotide sequence of pegRNA plasmid used is also shown in SEQ ID NO. 2. For different inversion events, the spacer sequence at bits 3501-3520 and the RTT and PBS sequences at bits 3597-3639 are different. The details are shown in Table 5 below.

TABLE 5

② Transfection, amplification of target bands

The pegRNA plasmid and PE2 plasmid were co-transfected into HEK293T cells and the genomes were harvested after 3 days of culture. The occurrence of inversion event is detected by designing two pairs of primers at the interfaces of the two ends of the inversion region, respectively. Only the inverted sequence can amplify the band of interest. Specific primer sequences are shown in Table 6 below.

TABLE 6

As shown in the TAE gel electrophoresis chart of FIG. 10, ladder-shaped bands are found at the interfaces of the two ends of the amplified inverted sequence. The ladder was sequenced for one generation, as shown in FIG. 11, and amplification edits at the 10kb interface on chromosome 6 were verified.

2. Target DNA inversion method with PAM-in form of two pegRNA on the same side

(1) PIEv3 design

As shown in FIG. 9, the method is designed in such a way that two pegRNA on the same side are in the form of PAM-in, a complex consisting of nCas-RT and 4 pegRNA is targeted to four sites on the genome double-stranded DNA, and two pairs (four) of complementary 3' flaps are introduced by Reverse Transcriptase (RT) using Reverse Transcription Templates (RTT) which do not have homology with the genome sequence.

When the two complementary pairs of 3' flaps are annealed together, inversion is completed under the repair mechanism (yellow part in FIG. 9). The accuracy of the interfaces at the two ends is ensured by two pairs of complementary 3' flaps, and the design of the target DNA inversion method is PIEv. As shown in the gray area in fig. 9, the PIEv method is adopted while deleting the sequences (a and C, B and D) between the two nick on the same side, preventing the occurrence of the second editing.

(2) PIEv3 feasibility verification of PIEv

Using the PIEv design, this example inverted the 10kb, 100kb and 1Mb regions on chromosome 6 in 293T cells. The specific method comprises the following steps:

The nucleotide sequence of pegRNA plasmid used is also shown in SEQ ID NO. 2. For different inversion events, the spacer sequence at bits 3501-3520 and the RTT and PBS sequences at bits 3597-3639 are different. The details are shown in Table 7 below.

TABLE 7

The pegRNA plasmid and PE2 plasmid were co-transfected into HEK293T cells and the genomes were harvested after 3 days of culture. The occurrence of inversion event is detected by designing two pairs of primers at the interfaces of the two ends of the inversion region, respectively. Only the inverted sequence can amplify the band of interest. The primers used are shown in Table 8 below.

TABLE 8

Using the PIEv strategy, 10kb, 100kb, 1Mb, and 30Mb regions on chromosome 6 were inverted in HEK293T and K562 cells. As shown in FIG. 12, the single band was found at the interface of both ends after the inversion of amplification. As shown in FIG. 13, sequences at the left and right interfaces of the inversion of 1Mb on chromosome 6 were determined, and the accuracy of the interfaces at both ends was verified by first-generation sequencing.

As shown in FIGS. 14a and 14b, the second generation sequencing showed that the two-terminal interface purity was very high after inversion of the 10 kb to 30 Mb region in both cell lines, 95.9-99.0% and 94.8-98.8% in the 293T and K562 cell lines, respectively.

To determine the efficiency of specific inversion editing, this example accurately quantifies the efficiency of the two-terminal interface where inversion occurs in the 10 kb to 30Mb region using the ddPCR method. As shown in FIGS. 14c and 14d, the efficiency of inversion in HEK293T cells was 7.9-39.6% and the efficiencies of inversion of 100kb and 30Mb in K562 cell lines were 57.4% and 11.4%, respectively, allowing efficient editing in both cell lines.

Example 3 design and feasibility verification of PIEv3b

1. PIEv3b design

In the PIEv method of example 2, when four pegRNA are cloned on four plasmid vectors, respectively, there may be a case where plasmid distribution is not uniform after transfection of cells. In this case, as shown in fig. 15, PIEv editing results may be caused, resulting in inaccurate inversion.

In order to reduce PIEv1 editing results, as shown in fig. 16, the present example clones two pegRNA that are distal and target different DNA strands on the same vector, no editing occurs when only one vector enters the cell, and PIEv3 editing occurs when only two vectors enter the cell, we call PIEv b method.

2. PIEv3b verification of the inversion editing efficiency in different cell lines

To verify the universality of PIEv b, we performed inversion editing of the 10 kb-1 Mb region on chromosome 6, 19 and 20 in human Huh-7, HAP1, heLa and hESCs cells, and 8 kb-50 Mb region on chromosome 2 in mouse mESCs and haESCs cells.

For the Chr 6-Inv10 kb, chr 6-Inv100 kb and Chr 6-Inv1Mb pegRNA was used identical to the Chr6 Inv10 kb-1-PIEv3, chr6 Inv100 kb-PIEv3 and Chr6 Inv 1Mb-1-PIEv3, respectively, of example 2. The details are shown in Table 9 below.

TABLE 9

In all cell lines, the genome was harvested 3 days after co-transfecting the cells with pegRNA and PE2, and the editing efficiency was quantified using ddPCR. In the human Huh-7 cell line, the inversion editing efficiency was 6.0-21.3% (FIG. 17 a), in the HAP1 cell line, the efficiency was 6.2-26.1% (FIG. 17 b), in the HeLa cell line, the efficiency was 18.4-46% (FIG. 17 c), and in the hESCs cell line, the efficiency was 8.8-62.0% (FIG. 17 d). In the mouse mESCs cell line, the inversion editing efficiency was 9.1-84.0% (FIG. 17 e), and in the haESCs cell line, the editing efficiency was 2.5-47.3% (FIG. 17 f). PIEv3b showed good inversion editing efficiency in different cell lines, demonstrating its versatility.

In addition, PIEv b produced higher precision inversion editing efficiency and fewer byproducts than DSB-mediated inversion, while the inversion in the DSB-produced editing product was only a small fraction, mostly DSB-induced random indels. PIEv3b exhibits higher editing efficiency than integrase-mediated inversion, especially over Mb-over genomic inversion, integrase is less efficient or no editing occurs.

3. PIEv3b edit completion verification

To further demonstrate the inversion editing ability of the PIEv b method, we performed a functional recovery test of GFP gene using PIEv b. The specific method comprises the following steps:

(1) A GFP expression plasmid with reverse inactivity was constructed and flanked by four pegRNA targeting sites (A, B, C and D). The sequence of GFP expression plasmid with reverse inactivity is shown as SEQ ID NO. 116.

(2) It was integrated into 293T cells with lentiviruses to construct stable expression cell lines.

In the cell line which was not edited, normal expression was not possible due to the reverse presence of GFP gene. After inversion editing by PIEv b method, GFP gene was inverted to return to normal, and fluorescence was generated, as shown in FIG. 18 a. As shown in fig. 18b, 13.1% of the cells were edited by flow sorting, restoring GFP expression.

For GFP-recovery, pegRNA was used as shown in SEQ ID NO. 2. For different inversion events, the spacer sequence at bits 3501-3520 and the RTT and PBS sequences at bits 3597-3639 are different. The details are shown in table 10 below.

Table 10

The present example also uses PIEv b method to invert the 40Mb region on chromosome 6 in HEK293T cells and verify the integrity. For Chr6-Inv 40Mb, pegRNA was used as shown in SEQ ID No. 2. For different inversion events, the spacer sequence at bits 3501-3520 and the RTT and PBS sequences at bits 3597-3639 are different. The details are shown in Table 11 below.

TABLE 11

After 293T cells are transfected, single cell clone sorting is carried out, and after the cells grow up, the cells are identified. And carrying out fluorescence in situ hybridization test on the identified positive single cell clone subjected to inversion editing. The probe used in this example contains two kinds of fluorescence, green and red, the green fluorescence marks the centromere of chromosome 6, and the red fluorescence marks the ESR1 gene at the end of the long arm of chromosome 6.

As shown in FIG. 19, in the cells not edited, the red fluorescence is located at the end of the long arm of chromosome 6, and in the cells in which inversion editing has occurred, the red fluorescence is located at the middle position of the long arm of chromosome 6. This demonstrates that the 40Mb region is inverted.

Example 4 use of the DNA guide editing System of the invention in the treatment of hemophilia A

1. Use of disease-associated sites

Some diseases and cancers are associated with inversion of large fragments on the genome. Hemophilia A (HA) is produced by inactivation of the F8 gene by recombination between inverted repeats on the X chromosome and can be classified into Inv1 (140 kb) and Inv22 (550 kb) depending on the position of inversion. In addition, some inversions may occur to cause gene fusion at a far distance, forming oncogenes. Such as Acute Myeloid Leukemia (AML) is produced by the inverted formation of the CBFB-MYH11 oncogene at 52 Mb on chromosome 16. In HEK293T cells we used PIEv b to perform inversion editing of 140 kb and 550 kb on the X chromosome while deleting the reverse homology that caused the disease inversion, we also performed inversion of 52 Mb on chromosome 16 to mimic AML genotype.

The nucleotide sequences of the pegRNA plasmids used are likewise shown in SEQ ID NO.2 for ChrX-Inv 140 kb, chrX-Inv 550 kb and Chr16-Inv 52 Mb. For different inversion events, the spacer sequence at bits 3501-3520 and the RTT and PBS sequences at bits 3597-3639 are different. As shown in table 12 below.

Table 12

The genome was harvested 3 days after co-transfection of pegRNA and PE2 into 293T cells, ddPCR identified that in the 140kb and 550kb inversions of hemophilia A we achieved an inversion editing efficiency of 21.2-31.5% and in the 52Mb inversions of Acute Myeloid Leukemia (AML) up to 15% (FIG. 20). These results indicate that PIEv b has great potential in creating disease models.

2. Creation of mutant with chromosomal Structure variation

During biological evolution, human chromosome structures exhibit either a central centromere or a sub-central centromere structure, whereas as mice with high similarity to the human genome, the chromosome structures all exhibit a consistent end centromere structure (except for the Y chromosome). This example uses the PIEv b method of example 3 to change the chromosome of a human cell from the "X" form of the centromere structure to the "V" form of the telomere structure, as shown in FIG. 21.

Specifically, the present example selects a 30Mb region on chromosome 20 and a 100Mb region on chromosome 2, respectively, for inversion. The nucleotide sequences of the pegRNA plasmids used for the Chr20-Inv 30Mb and the Chr2-Inv 100Mb are also shown in SEQ ID NO. 2. For different inversion events, the spacer sequence at bits 3501-3520 and the RTT and PBS sequences at bits 3597-3639 are different. The details are shown in Table 13 below.

TABLE 13

PegRNA and PE2 were co-transfected into 293T cells and then subjected to monoclonal sorting, followed by fluorescent in situ hybridization assays on positive clones identified as having inversion editing.

As shown in FIG. 22, in the probe targeting chromosome 2 used, the centromere of chromosome 2 was green-fluorescently labeled, and in the cells not edited, chromosome 2 exhibited the "X" shape of the central centromere structure, while in the cells where inversion editing occurred, chromosome 2 exhibited the "V" shape of the terminal centromere structure. This demonstrates that inversion of the 100Mb region occurs.

As shown in FIG. 23, the probe targeting chromosome 20 used contained two fluorescent portions of red and green, the red fluorescent labeled YWHAB gene on the long arm, the green fluorescent labeled MARPE gene near the centromere on the long arm, and the inverted terminal interface located in the green fluorescent labeled region, but did not destroy the MARPE gene.

As shown in FIG. 23, in the cells not edited, the 20 # chromosome showed two green spots and two red spots, and the chromosome was X-shaped with the centromere structure, and in the cells with inversion editing, the 20 # chromosome had four green spots and two red spots, and the chromosome was V-shaped with the centromere structure, demonstrating that inversion of the 30Mb region occurred.

Example 5 design and feasibility verification of PIES

Although PIEv b is capable of producing efficient inversion editing on different chromosomes, deletion of small fragments is introduced at the interface at both ends. To solve this problem, we have attempted to use a pair pegRNA to achieve seamless inversion editing, named PIES (PIE SEAMLESS versions).

As shown in fig. 24. Under the action of PE2 (nCas-RT) and two pegRNA (pegRNA E and pegRNA F), two 3 'single-stranded DNAs (Inversion editing version 1, 3' flap) are introduced by Reverse Transcriptase (RT) at two sites on the two strands of the targeted genomic double-stranded DNA, the 3 'flap generated by pegRNA E is complementary to the genomic sequence on the left side of cleavage site B, and the 3' flap generated by pegRNA F is complementary to the genomic sequence on the right side of cleavage site A.

Inversion occurs under the repair of DNA when the two 3' flaps are annealed together with their complementary genomic regions, respectively. pegRNA E and pegRNA F are PAM-out designs, and this target DNA inversion method design is named PIES-PAM-out.

2. Feasibility verification of PIES-PAM-out

To verify the feasibility of PIES-PAM-out, this example attempted a 100kb and 1Mb inversion on chromosome 6 and a 10Mb inversion on chromosome 2 in HEK293T and K562 cells.

PegRNA is also used as shown in SEQ ID NO. 2. For different inversion events, the spacer sequence at bits 3501-3520 and the RTT and PBS sequences at bits 3597-3639 are different. As shown in table 14 below.

TABLE 14

In both cell lines, the genome was harvested 3 days after co-transfecting the cells with pegRNA and PE2, and the editing efficiency was quantified using ddPCR. As shown in FIGS. 25 (a) and (b), the inversion editing efficiency in HEK293T and K562 cells was 0.5-10.0% and 0-6.6%, respectively.

To further verify its precise seamless inversion editing, two-generation sequencing was performed on the two-terminal interface, as shown in fig. 26, with product purity of 95.0-97.0% in HEK293T cells. It is verified that PIES-PAM-out can produce accurate seamless inversion editing.

The above detailed description describes in detail the practice of the invention, but the invention is not limited to the specific details of the above embodiments. Many simple modifications and variations of the technical solution of the present invention are possible within the scope of the claims and technical idea of the present invention, which simple modifications are all within the scope of the present invention.

Claims

1. A DNA leader editing system for inverting a DNA sequence of interest, wherein the DNA leader editing system comprises a Cas protein and a reverse transcriptase, pegRNA-A, and pegRNA-B;

The pegRNA-A comprises a first guide RNA, a first primer binding site and a first reverse transcription template, and the pegRNA-B comprises a second guide RNA, a second primer binding site and a second reverse transcription template, wherein the first guide RNA and the second guide RNA respectively target a target fragment;

2. The DNA leader editing system for inverting a target DNA sequence according to claim 1 wherein the sequence length of the first and second reverse transcription templates is 0 to 2,000 nucleotides, preferably 15 to 500 nucleotides.

3. The DNA leader editing system for inverting a target DNA sequence according to claim 1, wherein the first and second paired fragments have a sequence length of 0 to 1,000 nucleotides, preferably 3 to 200 nucleotides, more preferably 30 to 100 nucleotides.

4. The DNA leader editing system for reversing a DNA sequence of interest according to claim 1 wherein the first reverse transcription template further comprises 1 first non-complementary template sequence and the second reverse transcription template further comprises 1 second non-complementary template sequence, the first non-complementary template sequence and the second non-complementary template sequence being non-complementary to each other.

5. The system for DNA leader editing for inverted target DNA sequences according to claim 4, wherein the first and second non-complementary template sequences are 1 to 2,000 nucleotides in length, preferably 1 to 1,000 nucleotides in length, more preferably 1 to 500 nucleotides in length.

6. The DNA leader editing system for inverting a target DNA sequence according to claim 1 wherein the cleavage sites on either side of the same strand of double stranded DNA are 500 to 100,000,000 base pairs apart, preferably 10,000 to 30,000,000 base pairs apart.

7. The DNA leader editing system for inverted target DNA sequences according to claim 1 wherein the pegRNA-A and/or pegRNA-B further comprises a tail sequence which forms a hairpin or loop with itself or which comprises a poly (A), poly (U) or poly (C) sequence or which comprises an RNA binding domain.

8. A DNA leader editing system for inverting a target DNA sequence, wherein the DNA leader editing system comprises Cas protein and reverse transcriptase, pegRNA-A, pegRNA-B, pegRNA-C, pegRNA-D;

the pegRNA-A motif proximal to the protospacer sequence of the cleavage site that directs cleavage of the Cas protein is disposed proximal to or distal from the pegRNA-C motif proximal to the protospacer sequence of the cleavage site that directs cleavage of the Cas protein relative to the corresponding Spacer sequence.

9. The DNA leader editing system for inverting a target DNA sequence according to claim 8, wherein the sequence length between A and C is 2 to 10,000, preferably 500 to 2,000 nucleotides.

10. The DNA leader editing system for inverting a target DNA sequence according to claim 8, wherein the sequence length between B and D is 2 to 10,000, preferably 500 to 2,000 nucleotides.

11. The DNA leader editing system for inverting a target DNA sequence according to claim 8 wherein the cleavage sites flanking the same strand of the double stranded DNA are 500 to 100,000,000 base pairs apart, preferably 10,000 to 30,000,000 base pairs apart.

12. A DNA leader editing system for inverting a DNA sequence of interest, wherein the DNA leader editing system comprises a Cas protein and a reverse transcriptase, pegRNA-E, and pegRNA-F;

13. The DNA leader editing system for the inversion of a target DNA sequence according to claim 12 wherein the cleavage sites on both sides of the two opposite strands of double stranded DNA are 500 to 50,000,000 base pairs apart, preferably 1,000 to 500,000 base pairs apart.

14. A substance characterized in that it is any one of the following:

(1) A nucleic acid composition comprising a gene encoding the DNA leader editing system of any one of claims 1 to 13;

(2) A recombinant vector comprising a gene encoding the DNA leader editing system of any one of claims 1 to 13;

(3) A recombinant microorganism comprising a gene encoding the DNA leader editing system of any one of claims 1 to 13;

(4) A kit comprising the DNA leader editing system of any one of claims 1 to 13, or comprising the recombinant vector.

15. The substance of claim 14, wherein the recombinant vector comprises the coding gene of the DNA leader editing system of any one of claims 9-11, and wherein said pegRNA-A and pegRNA-D are recombined on the same recombinant vector, and wherein said pegRNA-B and pegRNA-C are recombined on the same recombinant vector.

16. Use of a DNA leader editing system as claimed in any one of claims 1 to 13 for inverting a target DNA sequence.

17. A method of inverting a target DNA sequence, comprising the steps of:

Adding the target DNA sequence and the DNA leader editing system as claimed in any one of claims 1 to 7 into a reaction system, and carrying out reverse transcription on the reverse transcriptase by using the first reverse transcription template and the second reverse transcription template respectively to generate 2 single-stranded DNA sequences, wherein the 2 single-stranded DNA sequences are complementarily combined to form a double-stranded region;

Or comprises the following steps:

Adding the target DNA sequence and the DNA leader editing system according to any one of claims 8-10 into a reaction system, wherein the reverse transcriptase respectively reverse transcribes the first reverse transcription template, the second reverse transcription template, the third reverse transcription template and the fourth reverse transcription template to generate 4 single-stranded DNA sequences, and the 4 single-stranded DNA sequences are complementarily combined to form 2 double-stranded regions;

Inverting the target DNA sequence based on a DNA repair mechanism and inserting a complementary DNA sequence of the first mating segment near the interface of the first mating segment and increasing in situ the sequence copy between A and C when the pegRNA-A motif adjacent to the original Spacer sequence of the cleavage site that directs cleavage of the Cas protein and the pegRNA-C motif adjacent to the original Spacer sequence of the cleavage site that directs cleavage of the Cas protein are disposed distally relative to the corresponding Spacer sequence;

Inverting the target DNA sequence based on a DNA repair mechanism when the primordial Spacer proximity motif of the cleavage site that directs cleavage of the Cas protein by pegRNA-A and the primordial Spacer proximity motif of the cleavage site that directs cleavage of the Cas protein by pegRNA-C are disposed in close proximity to the corresponding Spacer sequence, deleting a sequence copy between A and C and inserting a complementary DNA sequence of the first mating segment near the interface of the first mating segment;

Or comprises the following steps:

Adding the target DNA sequence and the DNA leader editing system as claimed in any one of claims 12 to 13 into a reaction system, carrying out reverse transcription by the reverse transcriptase by using the fifth reverse transcription template and the sixth reverse transcription template respectively to generate 2 single-stranded DNA sequences, combining the 2 single-stranded DNA sequences with corresponding complementary genome regions respectively to form double-stranded regions, and inverting the target DNA sequence based on a DNA repair action mechanism.

18. The method of claim 17, wherein the Cas protein is a Cas9 protein containing an inactivated HNH domain or a Cas9 protein containing an inactivated RuvC domain.

19. The method of claim 18, wherein the Cas protein is SpCas9、FnCas9、St1Cas9、St3Cas9、NmCas9、SaCas9、VQR SpCas9、EQR SpCas9、VRER SpCas9、SpCas9-NG、xSpCas9、RHA FnCas9、KKH SaCas9、NmeCas9、StCas9、CjCas9 or AtCas.

20. The method of claim 17, wherein the Cas protein is a Cas12 protein.

21. The method of claim 20, wherein the Cas12 protein is Cas12a, cas12b, cas12f or Cas12i.

22. The method of claim 21, wherein the Cas12 protein is one or more of AsCpf1、LbCpf1、FnCpf1、SsCpf1、PcCpf1、BpCpf1、CmtCpf1、LiCpf1、PmCpf1、Pb3310Cpf1、Pb4417Cpf1、BsCpf1、EeCpf1、BhCas12b、AkCas12b、EbCas12b、LsCas12b.