DNA pilot editing system of inversion target DNA sequence, recombinant vector, kit and method

Abstract

The invention discloses a DNA pilot editing system, a recombinant vector, a kit and a method for inverting a target DNA sequence, and relates to the technical field of gene editing. The DNA pilot editing system comprises a Cas protein, a reverse transcriptase, a pegRNA-A and a 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; the sequences of the first pairing fragment and the second pairing fragment are complementary; and the pegRNA-A and the pegRNA-B respectively guide the Cas protein to cut target fragments to be positioned at enzyme cutting sites on two sides of the same chain of the double-chain DNA. The invention provides four sets of DNA pilot editing systems based on the same technical concept. According to the four sets of DNA pilot editing systems, 3'single-stranded DNA is generated at the two ends of a target DNA sequence through specially designed pegRNA, and inversion editing of the target DNA is achieved through the complementary effect of paired fragments.

Description

Technical Field

[0001] The present invention relates to the field of gene editing technology, and in particular to a DNA lead editing system, a recombinant vector, a kit and a method for inverted target DNA sequences. Background Art

[0002] CRISPR-based genome editing tools have revolutionized our ability to manipulate genomic sequences with unprecedented precision. CRISPR-Cas nucleases induce site-specific double-strand breaks (DSBs) that enable targeted genome modifications through repair mechanisms such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). These pathways facilitate small insertions / deletions (indels) or precise modifications using donor templates.

[0003] In recent years, catalytically impaired Cas nucleases have been designed to collaborate with deaminases to form base editors (BEs), or with reverse transcriptases (RTs) to develop prime editors (PEs). Base editors allow efficient base replacement at specific genomic sites without relying on HDR or donor DNA. On the other hand, prime editors use prime editing guide RNAs (pegRNAs) to guide precise sequence modifications through RT templates encoded within the pegRNA. By combining PE with integrase, the range of precise DNA modification can be extended from a few base pairs (bp) to approximately 40 kb, a range that covers the average length of a single gene.

[0004] A large proportion of disease-causing mutations originate from structural variations in the genome. Although these variations can be induced by using the Cas9 nuclease in combination with a pair of guide RNAs (gRNAs), the major editing outcomes are usually small insertions / deletions, with structural variations being relatively minor byproducts. Despite the success of precise editing at single gene loci, the development of tools that can accurately and efficiently engineer large-scale structural variations in the genome of mammalian cells remains a critical unmet need.

[0005] Unlike deletions and duplications, inversions do not usually alter genomic copy number but can drastically affect gene expression and genome integrity. These rearrangements are critical for understanding human evolution and have been implicated in various genetic diseases, such as hemophilia A, mucopolysaccharidosis type II (Hunter syndrome), and Emory-Dreyfus muscular dystrophy, which are often caused by inversions between opposing homologous sequences. Furthermore, inversions can lead to the formation of oncogenic fusion genes, such as the ALK-EML4 fusion associated with lung cancer, underscoring 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 model chromosomal inversions in cells and animals. While inversions can be induced by Cas9 nuclease in combination with gRNA pairs, these events are uncommon and are often masked by indels. Recent approaches, such as combining PE with integrase and using two pairs of pegRNAs to install recombination sites, have demonstrated the ability to invert 40 kb fragments. However, these approaches remain inefficient for inversions in mammalian cells and require time-consuming serial transfections to install recombination sites prior to inversion. Given the critical role of inversions in genome structure and disease, there is an urgent need for more efficient and programmable tools to manipulate and study these structural variations. Summary of the invention

[0006] In view of the above shortcomings of the prior art, the present invention provides a DNA lead editing system, a recombinant vector, a kit and a method for inverted target DNA sequences, which utilizes lead editing guide RNA (pegRNA) to achieve inverted editing of target DNA sequences more accurately and efficiently. This is achieved specifically through the following technologies.

[0007] In a first aspect of the present invention, a first DNA lead editing system for inverting a target DNA sequence is provided, wherein the DNA lead editing system comprises a Cas protein and a reverse transcriptase, pegRNA-A and pegRNA-B;

[0008] 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;

[0009] 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 second paired fragment in sequence;

[0010] The pegRNA-A and the pegRNA-B respectively guide the Cas protein to cut the target fragment at the restriction sites on both sides of the same strand of the double-stranded DNA.

[0011] Furthermore, 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.

[0012] Furthermore, the sequence lengths of the first paired fragment and the second paired fragment are 0 to 1,000 nucleotides; preferably 3 to 200 nucleotides; more preferably 30 to 100 nucleotides.

[0013] Furthermore, the first reverse transcription template further comprises a first non-complementary template sequence, and the second reverse transcription template further comprises a second non-complementary template sequence; the first non-complementary template sequence and the second non-complementary template sequence are not complementary to each other.

[0014] Furthermore, the length of the first non-complementary template sequence and the second non-complementary template sequence is 1 to 2,000 nucleotides; preferably 1 to 1,000 nucleotides; more preferably 1 to 500 nucleotides.

[0015] Furthermore, the restriction sites located on both sides of 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.

[0016] Furthermore, the pegRNA-A and / or pegRNA-B also includes a tail sequence; the tail sequence forms a hairpin or a loop with itself, or the tail sequence includes a poly (A), poly (U) or poly (C) sequence, or the tail sequence includes an RNA binding domain.

[0017] The DNA lead editing system provided by the present invention introduces two complementary 3' flaps ( P rime editing-based I nversionwith E When these two complementary single-stranded DNA sequences are combined, an inversion occurs under the action of DNA repair ( Figure 1 ). This method is referred to as the PIEv1 method / design in this invention.

[0018] The second aspect of the present invention provides a second DNA lead editing system for inverting a target DNA sequence, characterized in that the DNA lead editing system comprises a Cas protein and a reverse transcriptase, pegRNA-A, pegRNA-B, pegRNA-C, and pegRNA-D;

[0019] 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; the pegRNA-D comprises a fourth guide RNA, a fourth primer binding site and a fourth reverse transcription template;

[0020] The first reverse transcription template includes a first paired fragment, the second reverse transcription template includes a second paired fragment, the third reverse transcription template includes a third paired fragment, and the fourth reverse transcription template includes a fourth paired fragment; the first paired fragment is complementary to the sequence of the second paired fragment, and the third paired fragment is complementary to the sequence of the fourth paired fragment;

[0021] The pegRNA-A and the pegRNA-B respectively guide the Cas protein to cut the target fragment at the restriction sites on both sides of one of the double-stranded DNA chains; the pegRNA-C and the pegRNA-D respectively guide the Cas protein to cut the target fragment at the restriction sites on both sides of the other chain of the double-stranded DNA chain;

[0022] The protospacer sequence adjacent motif of the restriction site where the pegRNA-A guides the Cas protein to cut and the protospacer sequence adjacent motif of the restriction site where the pegRNA-C guides the Cas protein to cut are set close to (PAM-in form) or away from (PAM-out form) relative to the Spacer sequence.

[0023] In the present invention, the PAM-out format is as follows Figure 7 shown. Figure 7 In the figure, the protospacer adjacent motif (PAM) of the restriction site of the Cas protein corresponding to pegRNA-A is located on the right side of the Spacer sequence (ProtospacerA); the protospacer adjacent motif (PAM) of the restriction site of the Cas protein corresponding to pegRNA-C is located on the left side of the Spacer sequence (ProtospacerB), and the two PAMs of pegRNA-A and pegRNA-C are set far away from each other relative to the two Spacer sequences. Similarly, the two PAMs of pegRNA-B and pegRNA-D are also set far away from each other relative to the two Spacer sequences.

[0024] In the present invention, the PAM-in form is as follows Fig. 9 shown. Fig. 9In the figure, the protospacer adjacent motif (PAM) of the restriction site of the Cas protein corresponding to pegRNA-A is located on the right side of the Spacer sequence (ProtospacerA); the protospacer adjacent motif (PAM) of the restriction site of the Cas protein corresponding to pegRNA-C is located on the left side of the Spacer sequence (ProtospacerB), and the two PAMs of pegRNA-A and pegRNA-C are close to each other relative to the two Spacer sequences. Similarly, the two PAMs of pegRNA-B and pegRNA-D are also close to each other relative to the two Spacer sequences.

[0025] Further, the length of the sequence between A and C is 2 to 10,000; preferably 500 to 2,000 nucleotides.

[0026] Further, the length of the sequence between B and D is 2 to 10,000; preferably 500 to 2,000 nucleotides.

[0027] Furthermore, the restriction sites located on both sides of the same strand of the double-stranded DNA are 500 to 100,000,000 base pairs apart;

[0028] More preferably, the distance between the two sequences is between 10,000 and 30,000,000 base pairs.

[0029] In order to develop precise inversion editing tools, we tried to introduce a second pair of complementary 3' single-stranded DNA on the other strand of the double-stranded DNA to ensure the accuracy of the interface at the other end. Figure 7 and 8 In the pegRNA-A and pegRNA-C, and pegRNA-B and pegRNA-D), there are two forms, PAM-out and PAM-in. "Adjacent / proximate" here refers to two pegRNAs located on two DNA chains and adjacent to each other. For example, pegRNA-A and pegRNA-C are defined as two "adjacent / proximate" groups of pegRNAs, and pegRNA-B and pegRNA-D are defined as two "adjacent / proximate" groups of pegRNAs. Therefore, for the two forms of PAM-out and PAM-in, the above-mentioned second DNA lead editing system provided by the present invention is actually divided into two more specific DNA lead editing systems, which are respectively named PIEv2 method / design and PIEv3 method / design in the present invention.

[0030] In the case of PAM-out, the PIEv2 method / design targets four sites on the genomic double-stranded DNA, and two pairs of complementary 3' single-stranded DNA are synthesized by reverse transcriptase RT. When these two pairs of complementary 3' single-stranded DNA are combined together, inversion occurs under the action of DNA repair. After the inversion editing is completed, the four original target sites still exist.

[0031] For the PAM-in form, the PIEv3 method / design also targets four sites on the genomic double-stranded DNA and synthesizes two pairs of complementary 3' single-stranded DNAs through RT. When these two pairs of complementary 3' single-stranded DNAs are combined together, inversion occurs under the action of DNA repair, and the sequence between the two nicks at both ends of the same side is deleted to prevent the occurrence of secondary editing.

[0032] Compared with the PIEv1 method / design, the PIEv2 method / design and the PIEv3 method / design can achieve inversion editing more accurately.

[0033] In a third aspect of the present invention, a DNA lead editing system for inverting a target DNA sequence is also provided, wherein the DNA lead editing system comprises a Cas protein and a reverse transcriptase, pegRNA-E and pegRNA-F;

[0034] 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; the fifth guide RNA and the sixth guide RNA respectively target the target fragment;

[0035] The fifth reverse transcription template includes a fifth paired fragment, and the sixth reverse transcription template includes a sixth paired fragment; the fifth paired fragment is complementary to a gene fragment sequence that is adjacent to the protospacer sequence of the restriction site guided by the sixth guide RNA for cutting the Cas protein and is close to the 3' end, and the sixth paired fragment is complementary to a gene fragment sequence that is adjacent to the protospacer sequence of the restriction site guided by the fifth guide RNA for cutting the Cas protein and is close to the 3' end;

[0036] The pegRNA-E and the pegRNA-F respectively guide the Cas protein to cut the restriction sites on both sides of the target fragment on the two opposite strands of the double-stranded DNA; the original spacer sequence adjacent motif of the restriction site where the pegRNA-E guides the Cas protein to cut and the original spacer sequence adjacent motif of the restriction site where the pegRNA-F guides the Cas protein to cut are set away from the corresponding Spacer sequence.

[0037] Furthermore, the restriction sites on both sides of the two opposite strands of the double-stranded DNA are 500 to 50,000,000 base pairs apart; preferably, 1,000 to 500,000 base pairs apart.

[0038] It can be known that in the above-mentioned several DNA guide 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 their corresponding target fragments. "First", "second", "third", "fourth", "fifth" and "sixth" are only used to distinguish and do not produce any special technical limitation. For different CRISPR / Cas gene editing systems, the guide RNA can be a specific sgRNA or crRNA.

[0039] For example, in the CRISPR / Cas9 gene editing system, the guide RNA is sgRNA; in the CRISPR / Cas12 or CRISPR / Cas13 gene editing system, the guide RNA is crRNA.

[0040] In a fourth aspect of the present invention, a substance is provided, which is any one of the following:

[0041] (1) A nucleic acid composition, comprising a gene encoding any one of the DNA prime editing systems provided above in the present invention;

[0042] (2) A recombinant vector comprising a gene encoding any one of the DNA prime editing systems provided above in the present invention;

[0043] (3) A recombinant microorganism, wherein the recombinant microorganism contains a gene encoding any one of the DNA prime editing systems provided above in the present invention;

[0044] (4) A kit, comprising any one of the DNA lead editing systems provided above in the present invention, or comprising the recombinant vector.

[0045] Furthermore, when the recombinant vector includes the coding gene of the DNA lead 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.

[0046] When using the PIEv1 method / design, PIEv2 method / design or PIEv3 method / design for practical application, pegRNAs are generally cloned on different vectors. When this vector construction method is used, there may be uneven distribution of plasmids after transfection of cells. Especially when using the PIEv3 method / design, the above phenomenon may lead to PIEv1 editing results and produce non-precise inversions.

[0047] In order to reduce the occurrence of the above problems, for the PIEv3 method / design, the present invention clones two pegRNAs (pegRNA-A and pegRNA-D) that are distal and target different DNA chains on the same vector, and clones the other two pegRNAs (pegRNA-B and pegRNA-C) on the same vector. When only one of these two vectors is present in the cell, no editing will occur. Only when both vectors are in the cell at the same time will PIEv3 editing occur.

[0048] A fifth aspect of the present invention provides an application of any of the above-mentioned DNA prime editing systems in an inverted target DNA sequence.

[0049] A sixth aspect of the present invention provides a method for inverting a target DNA sequence, comprising the following steps:

[0050] The target DNA sequence and any one of the DNA lead editing systems provided in the first aspect of the present invention are added to the reaction system, the reverse transcriptase uses the first reverse transcription template and the second reverse transcription template to reversely transcribe and generate two single-stranded DNA sequences, and the two single-stranded DNA sequences complementarily bind to form a double-stranded region; based on the DNA repair mechanism, the target DNA sequence is inverted, and the complementary DNA sequence of the first paired fragment is inserted at the interface close to the first paired fragment;

[0051] Alternatively, include the following steps:

[0052] The target DNA sequence and the DNA prime editing system according to the second aspect of the present invention are added to the reaction system; the reverse transcriptase uses the first reverse transcription template, the second reverse transcription template, the third reverse transcription template and the fourth reverse transcription template to reverse transcribe and generate four single-stranded DNA sequences, respectively, and the four single-stranded DNA sequences complementarily bind to form two double-stranded regions;

[0053] When the two PAMs (the protospacer adjacent motifs of the restriction site cut by the Cas protein) of pegRNA-A and pegRNA-C are in a form far away from each other relative to the two Spacer sequences (the same applies to pegRNA-B and pegRNA-D), that is, in a PAM-out form, the target DNA sequence is inverted based on the DNA repair mechanism, the complementary DNA sequence of the first paired fragment is inserted at the interface close to the first paired fragment, and the sequence copy between A and C is increased in situ; the complementary DNA sequence of the third paired fragment is inserted at the interface close to the third paired fragment, and the sequence copy between B and D is increased in situ;

[0054] When the two PAMs (the protospacer adjacent motifs of the restriction site cut by the Cas protein) of pegRNA-A and pegRNA-C are close to each other relative to the two Spacer sequences (the same applies to pegRNA-B and pegRNA-D), and are in the PAM-in form, the target DNA sequence is inverted based on the DNA repair mechanism, and the sequence copy between A and C is deleted at the interface close to the first paired fragment, and the complementary DNA sequence of the first paired fragment is inserted; the sequence copy between B and D is deleted at the interface close to the third paired fragment, and the complementary DNA sequence of the third paired fragment is inserted;

[0055] Alternatively, include the following steps:

[0056] The target DNA sequence and the DNA lead editing system provided in the third aspect are added to the reaction system; the reverse transcriptase uses the fifth reverse transcription template and the sixth reverse transcription template to reversely transcribe and generate two single-stranded DNA sequences, respectively, and the two single-stranded DNA sequences are respectively combined with corresponding complementary genomic regions to form double-stranded regions; the target DNA sequence is inverted based on the DNA repair mechanism.

[0057] Furthermore, in the above-mentioned method for 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.

[0058] Furthermore, in the above-mentioned method of inverting the 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 AtCas9.

[0059] Furthermore, in the above-mentioned method for inverting the target DNA sequence, the Cas protein is Cas12 protein.

[0060] Furthermore, in the above-mentioned method of inverting the target DNA sequence, the Cas12 protein is Cas12a, Cas12b, Cas12f or Cas12i.

[0061] Specifically, optionally, in the above-mentioned 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, and LsCas12b.

[0062] Optionally, the target DNA sequence is located within a cell.

[0063] Optionally, the cell is a dividing cell or a non-dividing cell.

[0064] Optionally, the target DNA sequence is a telomere or a fragment thereof.

[0065] Optionally, the above method for inverting a target DNA sequence is performed in vitro or in vivo.

[0066] Optionally, each pegRNA includes a first guide RNA or a second guide RNA, a first primer binding site or a second primer binding site, a first reverse transcription template or a second reverse transcription template, arranged in a "5' to 3'" or "3' to 5'" direction.

[0067] Optionally, the reverse transcriptase is M-MLV reverse transcriptase or a reverse transcriptase capable of functioning under physiological conditions.

[0068] Optionally, the Cas protein and reverse transcriptase are provided indirectly as nucleic acid fragments encoding the corresponding proteins, or are provided directly as proteins.

[0069] Optionally, each pegRNA is provided indirectly as a recombinant DNA encoding said pegRNA, or directly as an RNA molecule.

[0070] Compared with the prior art, the present invention is beneficial in that it provides four sets of DNA lead editing systems based on the same technical concept. These four sets of DNA lead editing systems all generate complementary paired fragments at both ends of the same chain of the target DNA sequence through specially designed lead editing guide RNA (pegRNA), and achieve inversion editing of the target DNA through the complementary action of the paired fragments.

[0071] The four sets of DNA lead editing systems provided by the present invention can be used for any target DNA sequence that needs inversion editing, and are not limited to a specific species or a certain type of species. In practical applications, for the corresponding species, as long as the technical concept of the present invention is used to construct four sets of DNA lead editing systems, good gene inversion editing effects can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Schematic diagram of the process of achieving target DNA inversion using the method of PIEv1 in Example 1. The PE2-pegRNA complex targets two sites on the same strand of the genomic double-stranded DNA. Two complementary 3' single-stranded DNAs are synthesized by RT using RTT that has no homology to the genomic sequence. When the two complementary 3' single-stranded DNAs are annealed together, inversion occurs under the action of DNA repair (yellow part).

[0073] Figure 2 This is a TAE gel electrophoresis diagram of the inversion detection results in Example 1; 10 kb, 100 kb and 1 Mb inversions on chromosome 6 were confirmed.

[0074] Figure 3 The first generation sequencing (Sanger sequencing) results of the interface sequence after the inversion in Example 1 determined the left and right interface sequences of the 1Mb inversion on chromosome 6, with the left side being precise and the right side having random insertions and deletions.

[0075] Figure 4 and Figure 5 This is the verification result of the integrity of the inversion in Example 1. Figure 4 Sanger sequencing confirmed the wild-type genome in HEK293T cells. Figure 5 It was verified that an 836 bp inversion occurred at the VEGFA site.

[0076] Figure 6 This is the inversion editing efficiency of PIEv1 in Example 1 in HEK293T and N2a cells.

[0077] Figure 7 Schematic diagram of the process of achieving target DNA inversion using the PIEv2 method of Example 2. The PE2-pegRNA complex targets four sites on the genomic double-stranded DNA. Two pairs of complementary 3' single-stranded DNAs are synthesized by RT. When these two pairs of complementary 3' single-stranded DNAs are combined together, inversion occurs under the action of DNA repair (yellow part). After the inversion editing is completed, the four target sites still exist.

[0078] Figure 8Schematic diagram of secondary editing after PIEv2 inversion editing in Example 2; Amplification editing (AE) occurs on genomic DNA edited by PIEv2. When inversion editing is completed, the four original target sites still exist, and then two pairs of complementary 3' single-stranded DNA are resynthesized on different strands of the double-stranded DNA, inducing AE.

[0079] Fig. 9 Schematic diagram of the process of achieving target DNA inversion using the method of PIEv3 in Example 2. The PE2-pegRNA complex targets four sites on the genomic double-stranded DNA. Two pairs of complementary 3' single-stranded DNAs are synthesized by RT. When the two pairs of complementary 3' single-stranded DNAs are combined together, inversion occurs under the action of DNA repair, and partial fragments at both ends (gray parts) are deleted.

[0080] Fig.10 This is a TAE gel electrophoresis diagram of the inversion results detected by the PIEv2 method in Example 2; the 11 kb and 1 Mb inversions on chromosome 6 were confirmed.

[0081] Fig.11 This is the first-generation sequencing (Sanger sequencing) result of the interface sequence after inversion using the PIEv2 method in Example 2; the amplification editing after the 10kb inversion on chromosome 6 was determined.

[0082] Fig.12 This is a TAE gel electrophoresis diagram of the inversion results detected by the PIEv3 method in Example 2; 10 kb, 100 kb and 1 Mb inversions on chromosome 6 were determined.

[0083] Fig.13 This is the first-generation sequencing (Sanger sequencing) result of the interface sequence after inversion using the PIEv3 method in Example 2; the left and right interface sequences of the 1 Mb inversion on chromosome 6 were determined, and both the left and right sides were accurate.

[0084] Fig.14 For Example 2, HEK293T ( Fig.14 a) and K562 ( Fig.14 b) Product purity of the interface sequences at both ends of the inversion region from 10 kb to 30 Mb on chromosome 6 in cells, and HEK293T ( Fig.14 c) and K562 ( Fig.14 d) Editing efficiency of inversions in regions ranging from 10 kb to 30 Mb on chromosome 6 in cells, quantified by ddPCR.

[0085] Fig.15 and 16 Schematic diagram of PIEv3b reducing imprecise editing in Example 3.

[0086] Fig.17 This is the result of verifying the inversion editing efficiency of PIEv3b in different cell lines in Example 3.

[0087] Fig.18 For Example 3, a functional cell line was generated by targeted inversion at the EGFP locus. Fig.18 a is the design diagram of the cell line in which GFP function was restored. Fig.18 b Editing efficiency of 957 bp inversion in the reverse inactivation region of EGFP, quantified by flow cytometry.

[0088] Fig.19 This is the intra-arm inversion event on chromosome 6 determined by fluorescent in situ hybridization in Example 3. Illustration of fluorescent in situ hybridization (FISH) of the 40 Mb inversion on chromosome 6. The first column from the left represents the centromere of chromosome 6 (green). In the second column, the red fluorescent probe labeled the approximately 198 kb genomic sequence around the ESR1 gene. Columns 1 and 2 are merged in column 3, and the nuclei are represented in blue. Column 4 is an enlarged view of column 3. Edit-1 and Edit-2 are different HEK293T single cell clones that underwent a 40 Mb inversion.

[0089] Fig. 20 This is the inversion editing effect of the DNA prime editing system of Example 4 on the hemophilia A and acute myeloid leukemia (AML) sites in HEK293T cells.

[0090] Fig.21 This is a schematic diagram of a human chromosome with an end centromere structure created by IE technology in Example 4.

[0091] Fig. 22 In Example 4, FISH was used to detect a 100 Mb inversion on Chr 2. Column 1 (starting from the left) shows the Chr 2 centromeric satellite DNA region labeled in green. Column 2 merges column 1 and DAPI staining, with the nuclei represented in blue. Column 3 provides a magnified view of column 2. Edit-1 and Edit-2 are different single cell clones from IE-treated samples for a 100 Mb inversion on Chr 2 in HEK293T cells.

[0092] Fig.23Detection of a 30 Mb inversion on Chr 20 using FISH. Column 1 (starting from the left) shows ~730 kb of genomic sequence near the MARPE1 gene labeled with a green fluorescent probe. Column 2 shows ~379 kb of genomic sequence near the YWHAB gene labeled with a red fluorescent probe. Columns 1 and 2 are merged in column 3, with nuclei stained blue. Column 4 is a magnification of column 3, and column 5 shows a magnification of DAPI staining only. Edit-1 and Edit-2 represent different single cell clones from IE-treated samples with a 30 Mb inversion on Chr 20 in HEK293T cells.

[0093] Fig.24 This is a schematic diagram of the design of the PIES-PAM-out method in Example 5 to achieve target DNA inversion.

[0094] Fig.25 The diagrams show the efficiency of inversion of 100 kb and 1 Mb on chromosome 6 and 10 Mb on chromosome 2 in HEK293T (a) and K562 (b) cells in Example 5.

[0095] Fig.26 The purity statistics of the interfaces at both ends after 100 kb and 1 Mb inversion on chromosome 6 in HEK293T cells in Example 5 are shown. DETAILED DESCRIPTION

[0096] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0097] It should be emphasized that the following specific implementation methods are only examples of the four sets of DNA pilot editing systems provided by the present invention to achieve gene inversion editing; they are not only for HEK293T cells, and the corresponding inversion editing effects can be obtained by using the following specific DNA pilot editing systems.

[0098] The four sets of DNA lead editing systems of the present invention are not limited to the specific Cas protein, reverse transcriptase or pegRNA sequence in the following specific embodiments, and the target DNA sequence is not limited to a specific species or a class of species. For any target DNA sequence that requires inversion editing, it is not limited to a specific species or a class of species. In practical applications, for the corresponding species, as long as the technical concept of the present invention is used to construct four sets of DNA lead editing systems, good gene inversion editing effects can be obtained.

[0099] Example 1: Design and feasibility verification of PIEv1

[0100] In order to achieve accurate and efficient inversion editing of the target DNA, this example uses a complex of PE2 and two pegRNAs to perform inversion editing of the target DNA.

[0101] 1. Design of PIEv1

[0102] like Figure 1 A complex consisting of PE2 (nCas9-RT) and two pegRNAs (pegRNA A and pegRNA B) was used to target two sites on the same strand of the genomic double-stranded DNA, and a reverse transcription template (RTT) that was not homologous to the genomic sequence was used to introduce two complementary 3' single-stranded DNAs (Inversion editingversion 1, 3' flap) through reverse transcriptase (RT).

[0103] When the two complementary 3' flaps are annealed together, inversion occurs under the action of DNA repair ( Figure 1 The yellow part in the figure). This targeted DNA inversion method was designed and named PIEv1.

[0104] 2. Feasibility verification of PIEv1

[0105] In order to verify the feasibility of PIEv1, this example attempts to perform 10kb, 100kb and 1Mb inversions on chromosome 6 in HEK293T cells. The specific method is as follows:

[0106] The PE2 plasmid sequence used is shown in SEQ ID NO.1.

[0107] The nucleotide sequence of the pegRNA plasmid used is shown in SEQ ID NO.2, wherein positions 3253-3500 are the promoter sequence, positions 3501-3520 are the spacer sequence, positions 3521-3596 are the sgRNA backbone sequence, positions 3597-3639 are the RTT and PBS sequences, and positions 3640-3684 are the linker and evopreQ1 motif sequences; wherein n is a degenerate base, representing any one of A, T, C, and G.

[0108] For different inversion events, the spacer sequences at positions 3501-3520 and the RTT and PBS sequences at positions 3597-3639 are different, as shown in Table 1 below.

[0109] Table 1

[0110] HEK293T cells were co-transfected with pegRNA plasmid and PE2 plasmid, and the genome was collected after 3 days of culture. The occurrence of inversion events was detected by designing two pairs of primers at the interfaces at both ends of the inversion region. Only the inverted sequence can amplify the target band. The relevant primers are shown in Table 2 below.

[0111] Table 2

[0112] Test results such as Figure 2 As shown in the TAE gel electrophoresis diagram, in the genome without any plasmid transfection (WT group), no band could be amplified using the F1 / F2 or R1 / R2 primer pairs; in the genome where inversion occurred after editing, the target band could be amplified using the F1 / F2 or R1 / R2 primer pairs, indicating that 10 kb, 100 kb and 1 Mb inversions were achieved on chromosome 6 of HEK293T cells.

[0113] like Figure 3 As shown in the figure, by performing first-generation sequencing (Sanger sequencing) on ​​the interface sequence after inversion, it was found that the left interface had an insertion of a flap sequence, which ensured accuracy under the action of flap complementation, while the right interface had obvious mutations (Indel), specifically random insertions and deletions. This indicates that the PIEv1 method has certain non-precise editing.

[0114] To verify the integrity of the inversion, we performed an 836 bp inversion on the VEGFA gene on chromosome 6. After transfecting 293T cells, the genome was collected and the amplified inversion band was used to construct a T / A clone, which verified the 836 bp inversion at the VEGFA site ( Figure 4 ), the first generation sequencing (Sanger sequencing) verified the integrity of the inversion ( Figure 5 ). The nucleotide sequence of the pegRNA plasmid used is also shown in SEQ ID NO. 2. For different inversion events, the spacer sequence at positions 3501-3520 and the RTT and PBS sequences at positions 3597-3639 are different. The details are shown in Table 3 below.

[0115] Table 3

[0116] The amplification primer VEGFA-inv 836 bp-F used is: 5'-ctctttagccagagccgggg-3', as shown in SEQ ID NO.23.

[0117] The amplification primer VEGFA-inv 836 bp-R used is: 5'-ggtggagggggtcggggct-3', as shown in SEQ ID NO.24.

[0118] 3. Cell line validation of PIEv1

[0119] To determine the editing efficiency of PIEv1 in cell lines, we inverted different regions on different chromosomes in human HEK293T cells and mouse N2a cells. The pegRNA used in human HEK293T cells is also shown in SEQ ID NO.2. For different inversion events, the spacer sequence at positions 3501-3520 and the RTT and PBS sequences at positions 3597-3639 are different. In mouse N2a cells, the spacer sequences and RTT and PBS sequences of different inversion events are specifically shown in Table 4 below.

[0120] Table 4

[0121] The pegRNA plasmid and PE2 plasmid were co-transfected into cells and cultured for 3 days before the genome was harvested. ddPCR was used to identify the inversion efficiency at the interface. The results showed that the efficiency in HEK293T and N2a cells was 32.8-38.4% and 0.4-10.1%, respectively ( Figure 6 a and b), indicating that PIEv1 can achieve effective inversion editing, but its efficiency is low at the chromosome level (above 1 Mb).

[0122] Example 2: Design and feasibility verification of PIEv2 (PAM-in) and PIEv3 (PAM-out)

[0123] In order to achieve more accurate inversion editing of the target DNA in this example, 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 interface at the other end. In other words, in addition to the two pegRNAs (pegRNA A and pegRNA B) provided in Example 1, two more pegRNAs (pegRNA C and pegRNA D) were selected in this example.

[0124] like Figure 7 and 9 As shown, for the two pegRNAs on the same side, there is a PAM-in (such as Fig. 9 ) and PAM-out (as shown Figure 7 Two forms are available.

[0125] Figure 7In the figure, the protospacer adjacent motif (PAM) of the restriction site of the Cas protein corresponding to pegRNA-A is located on the right side of the Spacer sequence (ProtospacerA); the protospacer adjacent motif (PAM) of the restriction site of the Cas protein corresponding to pegRNA-C is located on the left side of the Spacer sequence (ProtospacerB), and the two PAMs of pegRNA-A and pegRNA-C are far away from each other relative to the two Spacer sequences. Similarly, the two PAMs of pegRNA-B and pegRNA-D are also far away from each other relative to the two Spacer sequences.

[0126] Fig. 9 In the figure, the protospacer adjacent motif (PAM) of the restriction site of the Cas protein corresponding to pegRNA-A is located on the right side of the Spacer sequence (ProtospacerA); the protospacer adjacent motif (PAM) of the restriction site of the Cas protein corresponding to pegRNA-C is located on the left side of the Spacer sequence (ProtospacerB), and the two PAMs of pegRNA-A and pegRNA-C are close to each other relative to the two Spacer sequences. Similarly, the two PAMs of pegRNA-B and pegRNA-D are also close to each other relative to the two Spacer sequences.

[0127] The following are the corresponding target DNA inversion methods designed in the form of PAM-in and PAM-out.

[0128] 1. Target DNA inversion method with two pegRNAs on the same side both in PAM-out form

[0129] (1) Design of PIEv2

[0130] like Figure 7 As shown, this method designs two pegRNAs on the same side in the PAM-out form. The complex composed of nCas9-RT and four pegRNAs targets four sites on the genomic double-stranded DNA, uses a reverse transcription template (RTT) that has no homology to the genomic sequence, and introduces two pairs (four) of complementary 3' flaps through reverse transcriptase (RT).

[0131] When the two complementary 3' flaps are annealed and combined, the inversion is completed under the action of the repair mechanism ( Figure 7 The two pairs of complementary 3' flaps ensure the accuracy of the interfaces at both ends, and this target DNA inversion method is named PIEv2.

[0132] like Figure 8 Figure 2 shows the amplification editing (AE) that occurs on genomic DNA after being edited by PIEv2. When the inversion editing is completed, the four original target sites (Protospacer-A, B, C, and D) still exist, and the positions of A and D are swapped. Subsequently, two pairs of complementary 3' single-stranded DNAs are resynthesized on both strands of the double-stranded DNA, and AE is performed on the two end interfaces after the inversion.

[0133] (2) Feasibility verification of PIEv2

[0134] Using the PIEv2 design, this example performed inversions in the 10 kb and 1 Mb regions on chromosome 6 in 293T cells. The specific method is as follows:

[0135] The nucleotide sequence of the pegRNA plasmid used is also shown in SEQ ID NO. 2. For different inversion events, the spacer sequence at positions 3501-3520 and the RTT and PBS sequences at positions 3597-3639 are different. The details are shown in Table 5 below.

[0136] Table 5

[0137] ②Transfection and amplification of target band

[0138] HEK293T cells were co-transfected with pegRNA plasmid and PE2 plasmid, and the genome was collected after 3 days of culture. The occurrence of inversion events was detected by designing two pairs of primers at the interfaces at both ends of the inversion region. Only the inverted sequence can amplify the target band. The specific primer sequences are shown in Table 6 below.

[0139] Table 6

[0140] Test results such as Fig.10 As shown in the TAE gel electrophoresis diagram, ladder-like bands were found at the interfaces of both ends of the sequence after amplification and inversion. The ladder-like bands were sequenced by first generation sequencing, as shown in Fig.11 As shown, expansion editing at the 10 kb interface on chromosome 6 was verified.

[0141] 2. Target DNA inversion method with two pegRNAs on the same side both in PAM-in form

[0142] (1) Design of PIEv3

[0143] like Fig. 9As shown, this method designs two pegRNAs on the same side in the PAM-in form. The complex composed of nCas9-RT and four pegRNAs targets four sites on the genomic double-stranded DNA, uses a reverse transcription template (RTT) that has no homology to the genomic sequence, and introduces two pairs (four) of complementary 3' flaps through reverse transcriptase (RT).

[0144] When the two complementary 3' flaps are annealed and combined, the inversion is completed under the action of the repair mechanism ( Fig. 9 The yellow part in the figure). Two pairs of complementary 3' flaps ensure the accuracy of the interfaces at both ends. This target DNA inversion method is named PIEv3. Fig. 9 As shown in the gray area in the figure, the PIEv3 method was used to simultaneously delete the sequences between the two nicks on the same side (A and C, B and D) to prevent secondary editing.

[0145] (2) Feasibility verification of PIEv3

[0146] Using the PIEv3 design, this example performed inversions in the 10kb, 100kb, and 1Mb regions on chromosome 6 in 293T cells. The specific method is as follows:

[0147] The nucleotide sequence of the pegRNA plasmid used is also shown in SEQ ID NO. 2. For different inversion events, the spacer sequence at positions 3501-3520 and the RTT and PBS sequences at positions 3597-3639 are different. The details are shown in Table 7 below.

[0148] Table 7

[0149] HEK293T cells were co-transfected with pegRNA plasmid and PE2 plasmid, and the genome was collected after 3 days of culture. The occurrence of the inversion event was detected by designing two pairs of primers at the interfaces of both ends of the inversion region. Only the inverted sequence can amplify the target band. The primers used are shown in Table 8 below.

[0150] Table 8

[0151] Using the PIEv3 strategy, 10 kb, 100 kb, 1 Mb, and 30 Mb regions on chromosome 6 were inverted in HEK293T and K562 cells. Fig.12 As shown, a single band was found at the interface of both ends after amplification inversion. Fig.13 As shown, the sequences at the left and right interfaces of the 1 Mb inversion on chromosome 6 were determined, and the accuracy of the interfaces at both ends was verified by first-generation sequencing.

[0152] like Fig.14 As shown in a and 14b, next-generation sequencing showed that the purity of the interfaces at both ends of the 10 kb to 30 Mb region after inversion in the two cell lines was very high, which was 95.9-99.0% and 94.8-98.8% in 293T and K562 cell lines, respectively.

[0153] In order to determine the efficiency of specific inversion editing, this example uses the ddPCR method to accurately quantify the efficiency of the interfaces at both ends of the inversion in the 10 kb to 30 Mb region. Fig.14 As shown in Figures c and 14d, efficient editing was achieved in both cell lines, with an inversion efficiency of 7.9-39.6% in HEK293T cells and 57.4% and 11.4% in inversion efficiencies of 100kb and 30Mb in K562 cell lines, respectively.

[0154] Example 3: Design and feasibility verification of PIEv3b

[0155] 1. Design of PIEv3b

[0156] In the PIEv3 method of Example 2, when four pegRNAs are cloned on four plasmid vectors respectively, there may be uneven distribution of plasmids after transfection of cells. Fig.15 As shown, this may result in PIEv1 editing results, producing non-precise inversions.

[0157] To reduce the PIEv1 editing results, such as Fig.16 As shown, in this embodiment, two pegRNAs that are distal and target different DNA chains are cloned on the same vector. When only one vector enters the cell, no editing will occur. Only when both vectors enter the cell will PIEv3 editing occur. We call this the PIEv3b method.

[0158] 2. Verification of the inversion editing efficiency of PIEv3b in different cell lines

[0159] To verify the universality of PIEv3b, we performed inversion editing on the 10 kb-1 Mb region on chromosomes 6, 19, and 20 in human Huh-7, HAP1, HeLa, and hESCs cells; and on the 8 kb-50 Mb region on chromosome 2 in mouse mESCs and haESCs cells.

[0160] For Chr 6-inv10 kb, Chr 6-inv100 kb and Chr 6-inv1Mb, the pegRNA used was the same as Chr6 Inv 10kb-1-PIEv3, Chr6 Inv 100kb-PIEv3 and Chr6 Inv 1Mb-1-PIEv3 in Example 2, respectively. The details are shown in Table 9 below.

[0161] Table 9

[0162]

[0163] In all cell lines, the genome was harvested 3 days after co-transfection of 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 HeLa cell lines, the efficiency was 18.4-46% ( Fig.17 c); In hESCs cell lines, the efficiency was 8.8-62.0% ( Fig.17 d). In mouse mESCs cell lines, the inversion editing efficiency was 9.1-84.0% ( Fig.17 e); In the haESCs cell line, the editing efficiency was 2.5-47.3% ( Fig.17 f). In different cell lines, PIEv3b showed good inversion editing efficiency, proving its universality.

[0164] In addition, compared with DSB-mediated inversions, PIEv3b produces higher precision inversion editing efficiency and fewer byproducts, while inversions account for only a small portion of the editing products generated by DSBs, and most are random Indels induced by DSBs. Compared with integrase-mediated inversions, PIEv3b exhibits higher editing efficiency, especially for genomic inversions above Mb, where integrase efficiency is very low or no editing occurs.

[0165] 3. PIEv3b Edit Integrity Verification

[0166] To further demonstrate the inversion editing ability of the PIEv3b method, we used PIEv3b to perform a functional restoration experiment on the GFP gene. The specific method is as follows:

[0167] (1) A GFP expression plasmid with reverse inactivity was constructed, and four pegRNA targeting sites (A, B, C and D) were attached to both sides of the plasmid. The sequence of the GFP expression plasmid with reverse inactivity is shown in SEQ ID NO.116.

[0168] (2) Use lentivirus to integrate it into 293T cells to construct a stable expression cell line.

[0169] In the unedited cell line, the GFP gene cannot be expressed normally because it exists in reverse. After inversion editing by the PIEv3b method, the GFP gene is reversed and restored to normal, thus producing fluorescence. Fig.18 As shown in a. Fig.18 As shown in b, flow sorting showed that 13.1% of the cells were edited and GFP expression was restored.

[0170] For GFP-recovery, the pegRNA used is also shown in SEQ ID NO. 2. For different inversion events, the spacer sequence at positions 3501-3520 and the RTT and PBS sequences at positions 3597-3639 are different. The details are shown in Table 10 below.

[0171] Table 10

[0172] This example also uses the PIEv3b method to perform an intra-arm inversion on a 40Mb region on chromosome 6 in HEK293T cells and verifies its integrity. For Chr6-Inv 40 Mb, the pegRNA used is also shown in SEQ ID NO.2. For different inversion events, the spacer sequence at positions 3501-3520 and the RTT and PBS sequences at positions 3597-3639 are different. The details are shown in Table 11 below.

[0173] Table 11

[0174] After transfection of 293T cells, single cell clones were sorted and identified after the cells grew up. Fluorescence in situ hybridization was performed on the positive single cell clones that were identified to have undergone inversion editing. The probe used in this example contains two types 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.

[0175] like Fig.19 As shown, in unedited cells, the red fluorescence is located at the end of the long arm of chromosome 6; in cells with inversion editing, the red fluorescence is located in the middle of the long arm of chromosome 6. This proves that the 40Mb region has been inverted.

[0176] Example 4: Application of the DNA lead editing system of the present invention in the treatment of hemophilia A

[0177] 1. Application of disease-related sites

[0178] Some diseases and cancers are associated with large inversions on the genome. Hemophilia A (HA) is caused by the inactivation of the F8 gene due to recombination between inverted repeat sequences on the X chromosome. Depending on the location of the inversion, it can be divided into Inv1 (140 kb) and Inv22 (550 kb). In addition, some inversions may cause gene fusions at a long distance to form oncogenes. For example, acute myeloid leukemia (AML) is caused by the inversion of 52 Mb on chromosome 16 to form the CBFB-MYH11 oncogene. In HEK293T cells, we used PIEv3b to edit 140 kb and 550 kb inversions on chromosome X and deleted the reverse homologous sequence that caused the disease inversion; we also performed a 52 Mb inversion on chromosome 16 to simulate the AML genotype.

[0179] For ChrX-Inv 140 kb, ChrX-Inv 550 kb and Chr16-Inv 52 Mb, the nucleotide sequence of the pegRNA plasmid used is also shown in SEQ ID NO. 2. For different inversion events, the spacer sequence at positions 3501-3520 and the RTT and PBS sequences at positions 3597-3639 are different. The details are shown in Table 12 below.

[0180] Table 12

[0181] The genome was collected 3 days after pegRNA and PE2 were co-transfected into 293T cells. ddPCR identified that we achieved an inversion editing efficiency of 21.2-31.5% in the 140kb and 550kb inversions of hemophilia A, and up to 15% in the 52Mb inversion of acute myeloid leukemia (AML). Fig. 20 ). These results suggest that PIEv3b has great potential in creating disease models.

[0182] 2. Creation of chromosome structural variation mutants

[0183] In the process of biological evolution, the structure of human chromosomes presents a centromere or subcentromere structure, while the chromosome structure of mice, which is highly similar to the human genome, presents a consistent terminal centromere structure (except for the Y chromosome). This example uses the PIEv3b method of Example 3 to change the chromosomes of human cells from the "X" type of the centromere structure to the "V" type of the terminal centromere structure, such as Fig.21 shown.

[0184] Specifically, this embodiment selects a 30Mb region on chromosome 20 and a 100Mb region on chromosome 2 for inversion. For Chr20-Inv 30 Mb and Chr2-Inv 100 Mb, the nucleotide sequence of the pegRNA plasmid used is also shown in SEQ ID NO.2. For different inversion events, the spacer sequence at positions 3501-3520 and the RTT and PBS sequences at positions 3597-3639 are different. The details are shown in Table 13 below.

[0185] Table 13

[0186] After co-transfection of pegRNA and PE2 into 293T cells, single clones were sorted, and then fluorescent in situ hybridization was performed on the positive clones identified to have undergone inversion editing.

[0187] like Fig. 22 As shown, in the probe targeting chromosome 2 used, green fluorescence marks the centromere of chromosome 2; in unedited cells, chromosome 2 presents an "X" type of central centromere structure, while in cells with inversion editing, chromosome 2 presents a "V" type of terminal centromere structure. This proves that the inversion of the 100Mb region has occurred.

[0188] like Fig.23 As shown, the probe targeting chromosome 20 used contains two parts of red and green fluorescence. The red fluorescence marks the YWHAB gene on the long arm, the green fluorescence marks the MARPE1 gene near the centromere on the long arm, and the interface at one end of the inversion is located in the green fluorescence marked area, but the MARPE1 gene is not destroyed.

[0189] like Fig.23 As shown, in unedited cells, two green fluorescent spots and two red fluorescent spots appeared on chromosome 20, and the chromosome was an "X" type with a central centromere structure; in cells where inversion editing occurred, there were four green fluorescent spots and two red fluorescent spots on chromosome 20, and the chromosome was a "V" type with a terminal centromere structure, proving that inversion occurred in the 30Mb region.

[0190] Example 5: Design and feasibility verification of PIES

[0191] Although PIEv3b can produce efficient inversion editing on different chromosomes, it also introduces small deletions at the interface of both ends. To solve this problem, we tried to use a pair of pegRNAs to achieve seamless inversion editing, named PIES (PIE Seamless versions).

[0192] In a third aspect of the present invention, a DNA lead editing system for inverting a target DNA sequence is also provided, wherein the DNA lead editing system comprises a Cas protein and a reverse transcriptase, pegRNA-E and pegRNA-F;

[0193] 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; the fifth guide RNA and the sixth guide RNA respectively target the target fragment;

[0194] The fifth reverse transcription template includes a fifth paired fragment, and the sixth reverse transcription template includes a sixth paired fragment; the fifth paired fragment is complementary to a gene fragment sequence that is adjacent to the protospacer sequence of the restriction site guided by the sixth guide RNA for cutting the Cas protein and is close to the 3' end, and the sixth paired fragment is complementary to a gene fragment sequence that is adjacent to the protospacer sequence of the restriction site guided by the fifth guide RNA for cutting the Cas protein and is close to the 3' end;

[0195] The pegRNA-E and the pegRNA-F respectively guide the Cas protein to cut the restriction sites on both sides of the target fragment on the two opposite strands of the double-stranded DNA; the original spacer sequence adjacent motif of the restriction site where the pegRNA-E guides the Cas protein to cut and the original spacer sequence adjacent motif of the restriction site where the pegRNA-F guides the Cas protein to cut are set away from the corresponding Spacer sequence.

[0196] like Fig.24 As shown. Under the action of PE2 (nCas9-RT) and two pegRNAs (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 produced by pegRNA E is complementary to the genomic sequence on the left side of the restriction site B, and the 3' flap produced by pegRNA F is complementary to the genomic sequence on the right side of the restriction site A.

[0197] When the two 3' flaps are annealed to their complementary genomic regions, inversion occurs under the action of DNA repair. pegRNA E and pegRNA F are PAM-out designs, and this target DNA inversion method design is named PIES-PAM-out.

[0198] 2. Feasibility verification of PIES-PAM-out

[0199] To verify the feasibility of PIES-PAM-out, this example attempted to perform 100 kb and 1 Mb inversions on chromosome 6 and 10 Mb inversion on chromosome 2 in HEK293T and K562 cells.

[0200] The pegRNA used is also shown in SEQ ID NO. 2. For different inversion events, the spacer sequence at positions 3501-3520 and the RTT and PBS sequences at positions 3597-3639 are different. The details are shown in Table 14 below.

[0201] Table 14

[0202] In both cell lines, the genome was harvested 3 days after co-transfection of pegRNA and PE2, and the editing efficiency was quantified using ddPCR. Fig.25 As shown in (a) and (b), the inversion editing efficiencies in HEK293T and K562 cells were 0.5-10.0% and 0-6.6%, respectively.

[0203] In order to further verify its precise and seamless inversion editing, the interfaces at both ends were subjected to second-generation sequencing, such as Fig.26 As shown, in HEK293T cells, the product purity was 95.0-97.0%, verifying that PIES-PAM-out can produce precise seamless inversion editing.

[0204] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A DNA prime editing system for inverting a target DNA sequence, characterized in that: The DNA lead editing system includes Cas protein and 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 guide RNA and the second guide RNA target the target fragment respectively; 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 second paired fragment in sequence; The pegRNA-A and the pegRNA-B respectively guide the Cas protein to cut the target fragment at the restriction sites on both sides of the same strand of the double-stranded DNA.

2. The DNA prime editing system for inverting a target DNA sequence according to claim 1, characterized in that: The sequence lengths of the first reverse transcription template and the second reverse transcription template are 0 to 2,000 nucleotides, preferably 15 to 500 nucleotides.

3. The DNA prime editing system for inverting a target DNA sequence according to claim 1, characterized in that: The sequence lengths of the first paired fragment and the second paired fragment are 0 to 1,000 nucleotides, preferably 3 to 200 nucleotides, and more preferably 30 to 100 nucleotides.

4. The DNA prime editing system for inverting a target DNA sequence according to claim 1, characterized in that: The first reverse transcription template further comprises a first non-complementary template sequence, and the second reverse transcription template further comprises a second non-complementary template sequence; the first non-complementary template sequence and the second non-complementary template sequence are not complementary to each other.

5. The DNA prime editing system for inverting a target DNA sequence according to claim 4, characterized in that: The length of the first non-complementary template sequence and the second non-complementary template sequence is 1 to 2,000 nucleotides, preferably 1 to 1,000 nucleotides, and more preferably 1 to 500 nucleotides.

6. The DNA prime editing system for inverting a target DNA sequence according to claim 1, characterized in that: The restriction sites on both sides of the same strand of the double-stranded DNA are 500 to 100,000,000 base pairs apart; preferably, they are 10,000 to 30,000,000 base pairs apart.

7. The DNA prime editing system for inverting a target DNA sequence according to claim 1, characterized in that: The pegRNA-A and / or pegRNA-B also include a tail sequence; the tail sequence forms a hairpin or a loop with itself, or the tail sequence includes a poly(A), poly(U) or poly(C) sequence, or the tail sequence includes an RNA binding domain.

8. A DNA prime editing system for inverting a target DNA sequence, characterized in that: The DNA lead editing system includes Cas protein and reverse transcriptase, pegRNA-A, pegRNA-B, pegRNA-C, and 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; the pegRNA-D comprises a fourth guide RNA, a fourth primer binding site and a fourth reverse transcription template; The first reverse transcription template includes a first paired fragment, the second reverse transcription template includes a second paired fragment, the third reverse transcription template includes a third paired fragment, and the fourth reverse transcription template includes a fourth paired fragment; the first paired fragment is complementary to the sequence of the second paired fragment, and the third paired fragment is complementary to the sequence of the fourth paired fragment; The pegRNA-A and the pegRNA-B respectively guide the Cas protein to cut the target fragment at the restriction sites on both sides of one of the double-stranded DNA chains; the pegRNA-C and the pegRNA-D respectively guide the Cas protein to cut the target fragment at the restriction sites on both sides of the other chain of the double-stranded DNA chain; The original spacer sequence adjacent motif of the restriction site where the pegRNA-A guides the Cas protein to cut and the original spacer sequence adjacent motif of the restriction site where the pegRNA-C guides the Cas protein to cut are set close to or far away from the corresponding Spacer sequence.

9. The DNA prime editing system for inverting a target DNA sequence according to claim 8, characterized in that: The length of the sequence between A and C is 2 to 10,000; preferably 500 to 2,000 nucleotides.

10. The DNA prime editing system for inverting a target DNA sequence according to claim 8, characterized in that: The length of the sequence between B and D is 2 to 10,000; preferably 500 to 2,000 nucleotides.

11. The DNA prime editing system for inverting a target DNA sequence according to claim 8, characterized in that: The restriction sites on both sides of the same strand of the double-stranded DNA are 500 to 100,000,000 base pairs apart; preferably, they are 10,000 to 30,000,000 base pairs apart.

12. A DNA prime editing system for inverting a target DNA sequence, characterized in that: The DNA lead editing system includes 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; the fifth guide RNA and the sixth guide RNA respectively target the target fragment; The fifth reverse transcription template includes a fifth paired fragment, and the sixth reverse transcription template includes a sixth paired fragment; the fifth paired fragment is complementary to a gene fragment sequence that is adjacent to the protospacer sequence of the restriction site guided by the sixth guide RNA for cutting the Cas protein and is close to the 3' end, and the sixth paired fragment is complementary to a gene fragment sequence that is adjacent to the protospacer sequence of the restriction site guided by the fifth guide RNA for cutting the Cas protein and is close to the 3' end; The pegRNA-E and the pegRNA-F respectively guide the Cas protein to cut the restriction sites on both sides of the target fragment on the two opposite strands of the double-stranded DNA; the original spacer sequence adjacent motif of the restriction site where the pegRNA-E guides the Cas protein to cut and the original spacer sequence adjacent motif of the restriction site where the pegRNA-F guides the Cas protein to cut are set away from the corresponding Spacer sequence.

13. The DNA prime editing system for inverting a target DNA sequence according to claim 12, characterized in that: The restriction sites on both sides of the two opposite strands of the double-stranded DNA are 500 to 50,000,000 base pairs apart; preferably, they are 1,000 to 500,000 base pairs apart.

14. A substance, characterized in that The substance is any of the following: (1) A nucleic acid composition comprising a gene encoding the DNA prime editing system according to any one of claims 1 to 13; (2) A recombinant vector comprising a gene encoding the DNA prime editing system according to any one of claims 1 to 13; (3) A recombinant microorganism comprising a gene encoding the DNA prime editing system according to any one of claims 1 to 13; （4） A kit comprising the DNA lead editing system according to any one of claims 1 to 13, or comprising the recombinant vector.

15. The substance according to claim 14, characterized in that The recombinant vector includes the coding gene of the DNA lead editing system according to any one of claims 9 to 11, and 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.

16. Use of the DNA prime editing system according to any one of claims 1 to 13 in an inverted target DNA sequence.

17. A method for inverting a target DNA sequence, characterized in that: The following steps are involved: The target DNA sequence and the DNA lead editing system according to any one of claims 1 to 7 are added to the reaction system, the reverse transcriptase uses the first reverse transcription template and the second reverse transcription template to reversely transcribe and generate two single-stranded DNA sequences, and the two single-stranded DNA sequences complementarily bind to form a double-stranded region; based on the DNA repair mechanism, the target DNA sequence is inverted, and the complementary DNA sequence of the first paired fragment is inserted at the interface close to the first paired fragment; Alternatively, include the following steps: The target DNA sequence and the DNA lead editing system according to any one of claims 8 to 10 are added to the reaction system; the reverse transcriptase uses the first reverse transcription template, the second reverse transcription template, the third reverse transcription template and the fourth reverse transcription template to reversely transcribe and generate four single-stranded DNA sequences, and the four single-stranded DNA sequences complementarily bind to form two double-stranded regions; When the protospacer sequence adjacent motif of the restriction site where the pegRNA-A guides the Cas protein to cut and the protospacer sequence adjacent motif of the restriction site where the pegRNA-C guides the Cas protein to cut are set away from the corresponding Spacer sequence, the target DNA sequence is inverted based on the DNA repair mechanism, the complementary DNA sequence of the first paired fragment is inserted at the interface close to the first paired fragment, and the sequence copy between A and C is increased in situ; the complementary DNA sequence of the third paired fragment is inserted at the interface close to the third paired fragment, and the sequence copy between B and D is increased in situ; When the protospacer sequence adjacent motif of the restriction site where the pegRNA-A guides the Cas protein to cut and the protospacer sequence adjacent motif of the restriction site where the pegRNA-C guides the Cas protein to cut are set close to the corresponding Spacer sequence, the target DNA sequence is inverted based on the DNA repair mechanism, and the sequence copy between A and C is deleted at the interface close to the first paired fragment and the complementary DNA sequence of the first paired fragment is inserted; the sequence copy between B and D is deleted at the interface close to the third paired fragment and the complementary DNA sequence of the third paired fragment is inserted; Alternatively, include the following steps: The target DNA sequence and the DNA lead editing system according to any one of claims 12-13 are added to the reaction system; the reverse transcriptase uses the fifth reverse transcription template and the sixth reverse transcription template to reversely transcribe and generate two single-stranded DNA sequences, respectively, and the two single-stranded DNA sequences are respectively combined with the corresponding complementary genomic regions to form double-stranded regions; the target DNA sequence is inverted based on the DNA repair mechanism.

18. The method for inverting a target DNA sequence according to claim 17, characterized in that: The Cas protein is a Cas9 protein containing an inactivated HNH domain, or a Cas9 protein containing an inactivated RuvC domain.

19. The method for inverting a target DNA sequence according to claim 18, characterized in that: The Cas protein is SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, VQR SpCas9, EQR SpCas9, VRERSpCas9, SpCas9-NG, xSpCas9, RHA FnCas9, KKH SaCas9, NmeCas9, StCas9, CjCas9 or AtCas9.

20. The method for inverting a target DNA sequence according to claim 17, characterized in that: The Cas protein is Cas12 protein.

21. The method for inverting a target DNA sequence according to claim 20, characterized in that: The Cas12 protein is Cas12a, Cas12b, Cas12f or Cas12i.

22. The method for inverting a target DNA sequence according to claim 21, characterized in that: The Cas12 protein is one or more of AsCpf1, LbCpf1, FnCpf1, SsCpf1, PcCpf1, BpCpf1, CmtCpf1, LiCpf1, PmCpf1, Pb3310Cpf1, Pb4417Cpf1, BsCpf1, EeCpf1, BhCas12b, AkCas12b, EbCas12b, and LsCas12b.

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