Targeted replication method for DNA (deoxyribonucleic acid) fragments

Abstract

The present disclosure provides compositions and methods for replication / amplification of target fragments, suitable for use in target DNA sequences, such as genomic sequences. The editing system employs a pair of pegRNAs, which binds a reverse transcriptase (RT) template contained in the pegRNAs through targeting sites on both sides of a target fragment to expand the target fragment. As the two RT templates at least comprise mutually complementary parts, the two RT templates can form a double-stranded region, a starting point is provided for DNA polymerase, a new chain of each chain of a target fragment is synthesized, and the target fragment is copied. The proceeding of this process will cause amplification of the target sequence. Alternatively, the process can also be implemented by means of pegRNA / sgRNA or a combination of sgRNA / sgRNA. In the case of a PAM outer site of sgRNA / sgRNA, an RT enzyme and a template are not required.

Description

background

[0001] Inserting nucleic acid fragments into target nucleic acids (such as genomic sequences) is a challenging task. Targeted transgenic integration is usually achieved by homologous recombination repair (HDR), which, however, relies on exogenous DNA donors and is ineffective in non-dividing cells. Homologous-independent targeted integration (HITI) strategies are cell cycle-independent, but their efficiency at the genome level is low (usually about 1-5%), and a mixture of multiple editing events is observed.

[0002] A CRISPR-based gene editing tool has recently been developed that connects reverse transcriptase (RT) to Cas9 nickase, called Prime editing (PE). Its RT template (RTT) is located at the 3' end of the Prime editing guide RNA (pegRNA), allowing precise modification of the cut site. Prime editing can achieve various types of base substitutions, insertions and deletions of small fragments without relying on donor DNA, and has great application potential in basic research and correction of genetic mutations associated with human diseases. However, so far, Prime editing has not been used to insert large DNA fragments.

[0003] On the other hand, copying a portion of the target nucleic acid poses another challenge. In Prime editing technology, the inserted portion is primed by the RT template. Therefore, the length to be copied is limited. In addition, a new RT template is required for each target fragment to be copied. Overview

[0004] DNA sequence replication has great potential in treating a variety of genetic diseases and has huge commercial value in industrial settings such as protein production. Currently, there is no technology that can site-specifically amplify fragments on a target DNA sequence, at least not based on the newly developed CRISPR technology.

[0005] The current inventors have developed a new method, called Amplification Editing (AE), which is able to specifically amplify a target fragment in a target DNA sequence, such as a genomic sequence. An example of the AE method is to use a pair of pegRNAs whose targeting sites are located on both sides of the target fragment, and to extend the target fragment using a reverse transcriptase (RT) template contained in the pegRNA. Since the sequences contained in the two RT templates are at least partially complementary, they are able to form a double-stranded region, which then serves as the starting point for DNA polymerase to synthesize a new chain, thereby achieving the replication of the target fragment. Simply put, n rounds of amplification can produce up to 2 nAt the same time, the sequence encoded by the RT template will be inserted between the repeated target fragments.

[0006] According to one embodiment, the present disclosure provides a method for replicating a target fragment in a target DNA sequence in the presence of a DNA polymerase, the method comprising a target DNA sequence and components interacting therewith: (a) a Cas protein and a reverse transcriptase; (b) a first Prime Editing guide RNA (pegRNA) comprising a first CRISPR RNA (crRNA) and a first reverse transcriptase (RT) template sequence; (c) a second pegRNA, which contains a second crRNA and a second RT template sequence; wherein (i) the first RT template sequence contains a first pairing fragment, (ii) the second RT template sequence contains a second pairing fragment, (iii) the first pairing fragment and the second pairing fragment are complementary to each other, and (iv) the first pegRNA and the second pegRNA guide the Cas protein to cut at two sites on both sides of the target fragment on the target DNA sequence, on the complementary strands, thereby allowing (1) the reverse transcriptase to extend the two opposite strands of the target fragment using the first and second RT template sequences to generate two flap DNA sequences, and (2) the two flap DNA sequences to form a double-stranded region, allowing the DNA polymerase to extend the double-stranded region to replicate the target fragment, thereby achieving replication of the target fragment and inserting an insert fragment between the two repeated target fragments, wherein one strand of the insert fragment contains the reverse complementary sequence of the first fragment, the first complementary pairing fragment and the second fragment.

[0007] In certain embodiments, the first pegRNA further comprises a first primer binding site (PBS) and a first spacer sequence, and the second pegRNA further comprises a second PBS and a second spacer sequence, so that the pegRNA can guide the Cas protein to two sites on both sides of the target fragment and initiate reverse transcription.

[0008] In certain embodiments, the first and second RT template sequences are each 0 to 2000 nucleotides in length, preferably 15 to 500 nucleotides in length.

[0009] In certain embodiments, the first and second complementary pairing fragments are each 0 to 1000 nucleotides in length, preferably 3 to 200 nucleotides or 3 to 50 nucleotides in length, and more preferably 30 to 100 nucleotides in length.

[0010] In certain embodiments, the first and second RT template sequences each further comprise a non-complementary template sequence that is non-complementary to each other, wherein each non-complementary template sequence is located between the corresponding complementary pairing fragment and the crRNA, or between the corresponding complementary pairing fragment and the PBS.

[0011] In certain embodiments, each non-complementary template sequence is 1 to 2000 nucleotides in length, preferably 1 to 1000 or 1 to 500 nucleotides in length.

[0012] In certain embodiments, the two sites on either side of the target fragment are 2 to 1,000,000,000 base pairs apart, preferably 10 to 5,000,000 base pairs apart.

[0013] In certain embodiments, each RT template sequence further comprises an additional sequence adjacent to the complementary pairing fragment, wherein the two additional sequences are complementary to the target DNA sequence and are at least partially complementary to each other.

[0014] The present disclosure also provides a method for replicating a target fragment in a target DNA sequence in the presence of a DNA polymerase, the method comprising a target DNA sequence and the following components that interact therewith: (a) a Cas protein; (b) a first single guide RNA (sgRNA) or a transcribed RNA (tracrRNA); (c) a second sgRNA or tracrRNA, wherein the first sgRNA or tracrRNA and the second sgRNA or tracrRNA both have complementarity with target site sequences on both sides of the target DNA sequence fragment, and the two target site sequences have at least partial complementarity, wherein the first sgRNA or tracrRNA binds to one strand of the first target site in the presence of the Cas protein and cuts the opposite strand, releasing the opposite strand as a first single-stranded DNA; and the second sgRNA or tracrRNA binds to one strand of the second target site in the presence of the Cas protein and cuts the opposite strand, releasing the opposite strand as a second single-stranded DNA; and the first single-stranded DNA binds to the second single-stranded DNA to form a double-stranded region, allowing the DNA polymerase to extend the double-stranded region to replicate the target sequence, thereby achieving sequence replication between the two target sites.

[0015] In certain embodiments, the partial complementary length comprises full complementarity of at least 3, 4, 5, 6, 7 or 8 consecutive nucleotides.

[0016] The present disclosure also provides a method for replicating a target fragment in a target DNA sequence in the presence of a DNA polymerase, the method comprising a target DNA sequence and the following components that interact therewith: (a) a Cas protein and a reverse transcriptase; (b) a prime editing guide RNA (pegRNA) comprising a first CRISPR RNA (crRNA) and a reverse transcriptase (RT) template sequence; (c) a single-stranded guide RNA (sgRNA) or a transcription RNA (tracrRNA), wherein (i) the RT template sequence comprises a fragment that can be used for complementary pairing, (ii) the pegRNA guides the Cas protein to cut on the opposite strand on one side of the target fragment on the target DNA sequence, thereby allowing the reverse transcriptase to extend the opposite strand of the target fragment using the RT template sequence to generate a single-stranded DNA sequence, and (iii) the sgRNA or tracrRNA guides the Cas protein to cut on the opposite strand on the other side of the target fragment on the target DNA sequence, thereby releasing the opposite strand as a second single-stranded DNA sequence; and the two single-stranded DNA sequences form a double-stranded region, enabling the DNA polymerase to extend the double-stranded region to replicate the target fragment, thereby achieving replication of the target fragment.

[0017] In certain embodiments, the target DNA sequence is present inside a cell. In certain embodiments, the cell is a dividing cell. In certain embodiments, the cell is in a non-dividing state.

[0018] In certain embodiments, the target segment is a telomere or a segment thereof. In certain embodiments, the method is performed in vitro. In certain embodiments, the method is performed in vivo.

[0019] In certain embodiments, the Cas protein is a nickase. In certain embodiments, each pegRNA comprises a first or second crRNA, a first or second complementary pairing fragment, a first or second fragment, and a first or second PBS from 5' to 3'. In certain embodiments, the nickase is a Cas9 protein with an inactive HNH domain that cuts the target strand.

[0020] In certain embodiments, the nickase is a nickase from SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpf1, LbCpf1, FnCpf1, VQR SpCas9, EQR SpCas9, VRER SpCas9, SpCas9-NG, xSpCas9, RHA FnCas9, KKH SaCas9, NmeCas9, StCas9, CjCas9, or atCas9.

[0021] In certain embodiments, the Cas protein is a Cas12 protein.

[0022] In certain embodiments, each pegRNA comprises, from the 3' to the 5' direction, a first or second crRNA, a first or second PBS, a first or second fragment, and a first or second pairing fragment.

[0023] In certain embodiments, the Cas12 protein is Cas12a, Cas12b, Cas12f or Cas12i.

[0024] In certain embodiments, the Cas12 protein is from a selected group including AsCpf1, FnCpf1, SsCpf1, PcCpf1, BpCpf1, CmtCpf1, LiCpf1, PmCpf1, Pb3310Cpf1, Pb4417Cpf1, BsCpf1, EeCpf1, BhCas12b, AkCas12b, EbCas12b, and LsCas12b.

[0025] In certain embodiments, the first pegRNA or the second pegRNA further comprises a (a) tail capable of forming a hairpin or loop structure with itself, a PBS, an RT template sequence, a crRNA or a combined structure, or (b) comprises a poly(A), poly(U) or poly(C) sequence or an RNA binding domain.

[0026] In certain embodiments, the reverse transcriptase is M-MLV reverse transcriptase or a reverse transcriptase that functions under physiological conditions.

[0027] In certain embodiments, the Cas protein and reverse transcriptase are each provided in the form of nucleotides encoding the corresponding protein, or as a protein.

[0028] In certain embodiments, each pegRNA is provided in the form of recombinant DNA encoding the pegRNA or as an RNA molecule.

[0029] In certain embodiments, the duplicated target segments and insert segments are further replicated. Brief description of the drawing

[0030] Figure 1 Overview of Amplification Editing. Schematic diagram of Amplification Editing (AE). Figure 1 a shows the structure of two pegRNAs with opposite PAM orientations (i.e., the PAM sequence in the genome is not within the two sites targeted by the spacer). Figure 1As shown in b, the target site is cut by nCas9-RT and reverse transcribed into two complementary 3' flap DNAs. These two 3' flap DNAs anneal to each other and serve as primers to initiate DNA replication. Subsequently, the original genomic DNA is unwound and a new DNA chain is synthesized. When the new DNA chain extension encounters the nick, the synthesis stops.

[0031] Figure 2 Experimental design and results for detecting AE results. a, Schematic diagram of three PCR methods for detecting amplification editing. b, TAE agarose gel PCR amplification products of the method in Figure a show that 178bp and 234bp sequences at the VEGFA and HEK3 loci in HEK293T cells were amplified and 20bp were inserted. c, Sanger sequencing was performed on the edited bands after Out-Out PCR indicated by red triangles in Figure b for the VEGFA and HEK3 loci.

[0032] Figure 3 The effects of flap DNA length and complementary length at different loci were investigated. ab, The effects of flap DNA length and complementary length of 10-100 bp on editing efficiency were quantified by droplet digital PCR (ddPCR) when the replication lengths of the VEGFA and C-MYC loci were 178, 1014, and 7919 bp in HEK293T cells. c, DNA replication efficiency when the 3' flap of the VEGFA locus was 30, 40, 50, 80, or 100 bp and the complementary length was 30 bp. d, ddPCR design was used to quantify AE efficiency. Two primer / probe designs for control (reference gene / sequence) detection: one targeting the same chromosome at least 5 Kb away from the replication region and the other targeting a different chromosome. Primers for editing product detection were designed as In-In PCR, and probes were designed to target either the replication sequence or the insertion sequence.

[0033] Figure 4 Effects of 3' flap GC content and multiple editing on the AE method. a, Replication mediated by RTT at 30%, 50%, or 80% GC content at VEGFA and C-MYC loci. Replication efficiency was determined by ddPCR. Mean ± SD, n = 3 independent biological replicates. b, AE efficiency when co-transfected with 2 or 3 pairs of pegRNAs targeting different regions, as determined by ddPCR.

[0034] Figure 5Junction purity generated by the AE method. a, Design of head-to-tail PCR. PCR products were determined by next-generation sequencing (NGS). b, Junction purity in the control group (WT) and the edited group during 178 bp or 234 bp amplification at the VEGFA and HEK3 loci as measured by NGS. c, Junction purity during replication of 178, 1014, and 7919 bp mediated by different flap lengths and complementary lengths as measured by NGS. d-e, Junction purity of replication mediated by different flaps and complements at the VEGFA (d) and C-MYC (e) loci in HEK293T cells as measured by NGS.

[0035] Figure 6 Comparison of AE efficiency at each locus in different cell lines. a-e, AE efficiency of different amplification lengths in HEK293T cells (a), Huh-7 cells (b), K562 cells (c), U2OS cells (d), and N2a cells (e). Mean ± standard deviation, n = 3 independent biological replicates.

[0036] Figure 7 Editing results of AE shown by TAE agarose gel electrophoresis in different cell lines. a-c, Amplification products of In-In and In-Out PCR shown by TAE agarose gel electrophoresis.

[0037] Figure 8 Amplification editing for short sequences. a, Design of 20 bp replication. The length between the two nicks is 20 bp, and the two spacer sequences partially overlap. b, Replication efficiency of short fragment sequences. c, Analysis of Out-Out PCR amplification products for 20 bp replication by NGS.

[0038] Fig. 9 Tandem repeats achieved by the AE method. a, Overview of two models for multiple rounds of replication by AE. Left: The newly transcribed 3' flap sequences are complementary to each other; they anneal to each other and then synthesize new DNA strands, resulting in 2 n repeated sequences after n rounds of replication. Right: When the 3' flap pairs with the inserted sequence and anneals, its product has [dup*(n - 1)+x] amplified sequences (x < n), resulting in fewer than 2 n repeated sequences after n rounds of replication. b, Out-Out PCR amplification editing products of different single-cell clones shown by TAE agarose gel. The mixed results shown in the same clone are indicated by triangles. c, Sanger sequencing corresponding to the PCR amplification products in (b).

[0039] Fig.10Editing efficiency upon restriction enzyme digestion was measured by ddPCR. a, Amplification efficiency of VEGFA-178 bp and RUNX1-1132 bp in K562 cells was quantified by ddPCR. b, Schematic diagram of restriction enzyme digestion of editing products. Without digestion, the tandem repeats of the editing product genome are in one droplet (left); after digestion, each repeat is in a different droplet (right). cf, Duplication efficiency of VEGFA and HEK3 loci in K562 (c, d) and HEK293T (e, f) cells was measured by ddPCR. Extracted genomic DNA was measured with or without restriction enzyme treatment. Mean ± SD, n = 3 independent biological replicates. ***, P ≤ 0.001.

[0040] Fig.11 Restoration of EGFP function and replication of HBA gene by AE. a, The GFP sequence was disrupted and 53 bp coding sequence was missing. GFP-L, N-terminus of GFP; GFP-R, C-terminus of GFP. b, Quantification of cells treated with AE by flow cytometry. c, Schematic diagram of generating α-thalassemia genotype and restoring normal genotype. Double-strand breaks were generated in HBA1 (α1) and HBA2 (α2) genes using Cas9 and two sgRNAs, resulting in a 3.7 kb deletion and a fused α gene (named αh), which is identical to the α1 gene. The disease genotype was corrected to the normal genotype by AE. d, The replication efficiency of αh was determined by ddPCR.

[0041] Fig.12 Enhanced expression level of miR-21 by AE. a, Amplification of the stem-loop region of miR-21 precursor (197 bp). b, Replication efficiency of miR-21 at days 3, 5, and 7 in HEK293T and K562 cells. c, Quantification of miR-21 expression level by RT-qPCR. d, Expression levels of miR-21-targeted genes determined by RT-qPCR. **, P ≤ 0.01; *, P ≤ 0.05. Mean ± SD, at least n = 3 independent biological replicates.

[0042] Fig.13Replication of large, mega- and chromosome-sized DNA in HEK293T cells. a, Replication of 30-100 Kb on chromosomes 6 and 9 determined by In-In PCR. Top: PCR products visualized by TAE agarose gel. Bottom: Schematic diagram of Sanger sequencing of In-In PCR products. b, Copy number of IKZF4, RBMS2, and NEMP1 genes in a 1 Mb repeat region on chromosome 12 was determined in control (WT) and single-cell clones, respectively. c, Quantification of 30-100 Kb replication efficiency on chromosomes 6 and 9 by ddPCR. d, Quantification of 1-3 Mb replication efficiency on chromosomes 6, 9, and 12 by ddPCR. e, 10-100 Mb replication efficiency on chromosomes 6 and 9. Mean ± SD, n = 3 independent biological replicates, applicable to cf. ***, P ≤ 0.001.

[0043] Fig.14 Visualization of chromosome-level duplication after AE in HEK293T cells. ab, Fluorescence in situ hybridization (FISH) targeting the 3Mb duplication on chromosome 12 (a) and the 100Mb duplication on chromosome 6 (b). The first column (starting from the left) represents the centromere of chromosome 12 (a) or 6 (b). In the second column, the fluorescent probe labeled a genomic sequence of approximately 108Kb adjacent to the STAT6 gene (a) or a genomic sequence of approximately 198Kb adjacent to the ESR1 gene (b). The first and second columns are merged in the third column. The fourth column is a magnified view of the third column. For (a) or (b), Edit-1 and Edit-2 are two different single-cell clones from AE-treated samples, targeting the 3Mb (a) or 100Mb (b) duplications, respectively. The length unit of the fourth column in b is micrometers.

[0044] Fig.15 Exploring replication efficiency with shorter complementary lengths. a, Replication efficiency when using 0-10 bp 3' flaps for VEGFA-178 bp amplification.

[0045] Fig.16Various PAM-out amplification editing methods. a, Schematic diagram of amplification editing with different combinations of pegRNAs and sgRNAs. From top to bottom, the first one (I) is the AE design we described above; the second one (II) is similar to the first one, but part of the 3' flap is complementary to the genomic sequence near the cut; the third one (III) applies one sgRNA and one pegRNA, in which the 3' flap generated by the pegRNA is complementary to the genomic sequence near the sgRNA cut; the bottom design (IV) is amplification by two sgRNAs and Cas9 nickase in the absence of RT enzyme, but the two cut sequences are complementary to each other. b, Efficiency of replication in HEK293T cells using pegRNA+sgRNA or paired pegRNAs at 0, 8, 30, and 38 bp complementarity. c, Quantification of adapter purity in (b) by NGS. d, TAE agarose gel visualization of In-In PCR amplification products of paired sgRNA-mediated replication at 4 bp or 8 bp complementarity in HEK293T cells. e, Quantification of adapter purity in (e) by NGS. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. Mean ± SD, n = 3 independent biological replicates. Detailed description definition

[0046] It should be noted that "a" or "an" entity refers to one or more of that entity; for example, "an antibody" is understood to mean one or more antibodies. Therefore, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0047] As used herein, the term "polypeptide" is intended to encompass both a single "polypeptide" and a plurality of "polypeptides", and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). "Polypeptide" refers to a chain formed by two or more amino acids, and does not refer to a product of a specific length. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to two or more amino acid chains are included in the definition of "polypeptide", and the term "polypeptide" can replace or interchange with any of the above terms. The term "polypeptide" also refers to post-expression modification products of polypeptides, including but not limited to glycosylation, acetylation, phosphorylation, amination, derivatization with known protecting / blocking groups, proteolytic cleavage or modification with non-natural amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It may be generated in any manner, including chemical synthesis.

[0048] When referring to a polynucleotide, the term "encoding" means that if it can be transcribed and / or translated to produce mRNA of the polypeptide and / or its fragments in its original state or by manipulation by methods widely known to those skilled in the art, then the polynucleotide is said to "encode" a polypeptide. The antisense strand is the complementary sequence of the nucleic acid, and the coding sequence can be deduced therefrom. Amplification

[0049] Prime editing (PE) is a genome editing technology by which the genome of an organism can be modified. Prime editing writes new genetic information directly into a target DNA site. It uses a fusion protein consisting of a catalytically impaired endonuclease (e.g., Cas9) with an engineered reverse transcriptase, and a primeediting guide RNA (pegRNA) that recognizes the target site and delivers new genetic information to replace the target DNA nucleotide. Prime editing mediates targeted insertions, deletions, and base transitions without the need for double-strand breaks (DSBs) or donor DNA templates.

[0050] pegRNA is able to recognize the target nucleotide sequence to be edited and encode new genetic information that replaces the target sequence. pegRNA consists of an extended single guide RNA (sgRNA) containing a primer binding site (PBS) and a reverse transcriptase (RT) template sequence. During the genome editing process, the primer binding site allows the 3' end of the cut DNA chain to hybridize with the pegRNA, while the RT template serves as a template for synthesizing the edited genetic information. In the sgRNA part, there is a guide sequence (spacer) that guides the prime editor to the target genomic site and an sgRNA framework. When the guide sequence binds to the target genomic sequence and dissociates the DNA double helix, the PBS binds to the other chain and initiates reverse transcription, using the RT template sequence as a template. The newly synthesized sequence (a "3' flap") binds to the target genomic site to form double-stranded DNA. The RT template can contain mutations or small insertions relative to the target genomic sequence, but needs to be substantially homologous to the target genomic sequence, because the newly synthesized DNA chain should still remain hybridized with one of the original target genomic sequences.

[0051] The inventors have designed and implemented a new technology that can effectively and specifically amplify a target fragment on a target DNA sequence, such as a genomic sequence. The exemplary method uses a pair of pegRNAs, both of which, due to their targeting sites located on both sides of the target fragment, extend the target fragment through a reverse transcriptase (RT) template contained in the pegRNA. Since the two RT templates contain at least complementary parts, they can form a double-stranded region, thereby serving as a starting point for DNA polymerase to synthesize a new strand of each strand of the target fragment, thereby replicating the target fragment.

[0052] This new editing technology is called "Amplification Editing" (AE). Figure 1 As shown. In an example AE procedure, two pegRNAs are used. They are usually in the PAM-out position. Each pegRNA includes a CRISPR RNA (crRNA or sgRNA), a reverse transcriptase (RT) template sequence, and a primer binding site (PBS). The PBS can be complementary to the guide sequence (or "spacer sequence") in the crRNA, but is usually a few nucleotides shorter. When the guide sequence binds to the target genomic sequence and dissociates the DNA double helix, the PBS can bind to the other chain and initiate reverse transcription, using the RT template sequence as a template.

[0053] Unlike conventional prime editing pegRNAs, in each of the two pegRNAs in the amplification editing system, the RT template sequence does not have to be homologous to the target genomic sequence. In certain embodiments, the RT template preferably has reduced or even no homology to the target genomic sequence.

[0054] Instead, the two RT templates share a complementary portion. Figure 1 As shown, in each pegRNA, the RT template includes two parts, a paired RT fragment and an RT fragment. The two paired (complementary) RT fragments have complementary sequences, so the DNA sequences reverse transcribed from them can pair with each other.

[0055] It should be noted that although complementary portions are required to form flap DNA sequences that can bind to each other, the RT templates do not necessarily contain non-complementary "RT fragments." In other words, in some embodiments, the two RT templates are completely complementary to each other.

[0056] When the guide sequence binds to the target genomic sequence and dissociates the DNA double helix, the PBS binds to the other strand and initiates reverse transcription, using the RT template sequence as a template. Figure 1As shown, the two pegRNAs are designed in a PAM-out design such that they cut at two positions flanking the target portion (step 110). The RT template then serves as a template for synthesizing single-stranded DNA, thereby introducing two single-stranded "flaps" that extend the sequence to be replicated in opposite directions (step 120). The direction of extension is ensured by the design of the pegRNA molecule.

[0057] Each flap contains a reverse transcription of an RT segment from the pegRNA and further includes a portion from the paired (complementary) RT segment. Due to their complementarity, these two distal segments can hybridize with each other to form a double-stranded region (step 130). This double-stranded region can then serve as a starting point for DNA polymerase.

[0058] Using the double-stranded region as a starting point and the single-stranded DNA of the genome as a template, a new DNA strand is synthesized, unwinding the original DNA between the two incisions (steps 140 - 150). Eventually, the target sequence is precisely replicated, with a small flap sequence (the sequence generated by the 3' flap) inserted in between.

[0059] As shown in Examples 1 - 2 ( Figure 2-3 ), this newly designed amplification editing system has high precision and high efficiency. In addition, this new editing technology is extremely powerful, with the flap sequence able to be as short as 10 nucleotides or as long as 100 nucleotides or more, without significantly affecting the editing efficiency (Example 2, Figure 3 ). AE is active in a variety of cell lines and at multiple genomic loci (Example 3, Figure 6-7 ). Moreover, AE is capable of replicating human genomic regions ranging from 20 bp to at least 100 megabases (Mb), which is quite unexpected considering the average size of a human chromosome is approximately 100 Mb. (Examples 2, Figure 3 , Figure 8 , Figure 13-14 ).

[0060] In another surprising finding, since each round of AE does not disrupt the flanking sequences containing the PAM sequence or the cleavage site, amplification can be repeated. As Fig. 9 shown and demonstrated in Figure 9-10 , the AE method is indeed able to continue amplifying the target fragment. When the newly transcribed 3' flaps are complementary to each other, they anneal to each other, followed by the synthesis of a new DNA strand, resulting in a duplication of 2 n after n rounds of replication. While when the 3' flap pairs and anneals with the inserted sequence, the resulting product has [dup*(n - 1)+x] amplified sequences (x < n), leading to a duplication number less than 2 n after n rounds of replication. Alternative designs of AE

[0061] At the same time, it is worth noting that replication can be achieved by other methods besides paired pegRNAs. When these pairs are arranged in a PAM-out orientation and share complementary sequences near the cleavage site to allow annealing to form a DNA synthesis primer, they achieve pegRNA / sgRNA ( Fig.16 a, III) or sgRNA / sgRNA ( Fig.16 a, IV) combination ( Figure 15-16 ). In addition, the 3' flaps can be partially complementary to each other and to the genomic sequence near the cleavage site induced by another pegRNA ( Fig.16 a, II). pegRNA+sgRNA (or tracrRNA)

[0062] In Example 7, it is proved that ( Figure 15-16 ), the AE system does not require two complementary flaps. Fig.16 a Design (III), the left assembly still contains an nCas9-RT protein with a pegRNA that can generate a 3' flap during reverse transcription of the RT-paired fragment. On the right, only a conventional sgRNA (or conventional tracrRNA, i.e., without RT template) is used, which can work with either a conventional nCas9 protein or the same nCas9-RT protein, but does not require its RT activity.

[0063] In this design, the single 3' flap generated on the left is complementary to the genomic sequence near the cleavage site on the right. As shown in Example 7, their complementarity can also initiate DNA unwinding and replication, resulting in the duplication of the sequence between the two cleavage sites. sgRNA+sgRNA (or tracrRNA+tracrRNA, sgRNA+tracrRNA, tracrRNA+sgRNA)

[0064] Another alternative design is to not create a 3' flap. Therefore, the entire system ( Fig.16 a(IV)) does not contain reverse transcriptase or pegRNA. On the left and right sides, conventional nCas9 / sgRNA (or tracrRNA) assembly was used, provided that the genomic sequences near the two cleavage sites are at least partially complementary.

[0065] In the absence of a single 3' flap, at each site of cleavage, the exposed non-target strand allows annealing of two complementary non-target genomic sequences, forming a primer for DNA synthesis, leading to the occurrence of DNA replication. Mixed pegRNA + mixed pegRNA

[0066] An alternative to design (I) is design (II) ( Fig.16 a). This design (II) still uses two nCas9-RTs and two pegRNA sequences. Unlike design (I), the pegRNAs of design (II) are partially complementary to each other and partially complementary to the genomic sequences near the opposite cleavage sites.

[0067] Essentially, in design (II), not only the 3' flap is able to initiate annealing, but also the adjacent genomic sequence can play a role, thus allowing the 3' flap sequence to be shorter.

[0068] Therefore, according to one embodiment of the present disclosure, the present disclosure provides a method for replicating a target fragment in a target DNA sequence in the presence of a DNA polymerase. In certain embodiments, the method involves interacting the target DNA sequence with the following components: (a) a Cas protein and a reverse transcriptase, (b) a first prime editing guide RNA (pegRNA), the pegRNA comprising a first CRISPR RNA (crRNA / sgRNA), a first reverse transcriptase (RT) template sequence, and (c) a second prime editing guide RNA (pegRNA), the pegRNA comprising a second crRNA / sgRNA and a second RT template sequence. In certain embodiments, the first pegRNA also comprises a first primer binding site (PBS) and a first spacer, and the second pegRNA also comprises a second PBS and a second spacer.

[0069] In certain embodiments, the first RT template sequence includes a first pairing fragment, the second RT template sequence includes a second pairing fragment, and the first pairing fragment and the second pairing fragment are complementary. Therefore, the first pegRNA and the second pegRNA can guide the Cas protein to cut the opposite strands of the targeting sequence on both sides of the target DNA. Subsequently, the reverse transcriptase extends the two opposite strands of the targeting sequence using the first and second RT sequences as templates, thereby generating two single-stranded flap DNA sequences.

[0070] In addition, the two single-stranded flap sequences can form a double-stranded region, so that the DNA polymerase can extend the double-stranded region to replicate each target fragment. Therefore, the target fragment is replicated. At the same time, an insert fragment is inserted between the two replicated target fragments, wherein the strand of one insert fragment contains the reverse complementary sequence of the first fragment, the first paired fragment, and the second fragment.

[0071] In certain embodiments, each RT template sequence further comprises an additional sequence adjacent to the paired fragment ("mixed pegRNA / mixed pegRNA"), wherein the two additional sequences are complementary to the target DNA sequence and are at least partially complementary to each other.

[0072] There is also a method for replicating a target region in a genome in the presence of a DNA polymerase, the method comprising the following components, and interacting with a target sequence: (a) a Cas protein, (b) a first guide RNA (sgRNA1), and (c) a second guide RNA (sgRNA2), wherein the first guide RNA and the second guide RNA respectively have complementary sequences on both sides of a spacer sequence in a target DNA sequence, and the two target sites have at least partial complementarity; wherein, under the action of the Cas protein, the first guide RNA binds to one strand of the target site, cuts the opposing strand, and releases the opposing strand to form a first single-stranded DNA; under the action of the Cas protein, the second guide RNA binds to the other strand of the target site, cuts the opposing strand, and releases the opposing strand to form a second single-stranded DNA; the two single-stranded DNAs bind to form a double-stranded region, allowing the DNA polymerase to replicate the double-stranded region, thereby replicating the two strands between the target sequences, and ultimately replicating the target sequence.

[0073] In these methods, partial complementarity includes complete complementarity of at least 2, 3, 4, 5, 6, 7 or 8 nucleotides, which nucleotides are generally contiguous.

[0074] Also provided is a method for replicating a targeted region in a genome in the presence of a DNA polymerase, the method comprising the following components, and interacting with a target sequence: (a) a Cas protein and a reverse transcriptase, (b) a guide RNA (pegRNA), the pegRNA consisting of a CRISPR RNA (crRNA) and a reverse transcriptase (RT) template sequence, and (c) a single-stranded guide RNA (sgRNA); wherein: (i) the reverse transcriptase template sequence comprises a segment that can be paired, (ii) the pegRNA guides the Cas protein to cut on the targeted DNA, so that the reverse transcriptase uses the RT sequence as a template to extend the opposite strand of the target segment, thereby generating a single-stranded DNA sequence, and (iii) the sgRNA guides the Cas protein to cut on the opposite strand of the targeted sequence, releasing a second single-stranded DNA sequence; wherein the two single-stranded DNA sequences combine to form a double-stranded region, allowing the DNA polymerase to replicate the double-stranded region, thereby replicating each strand between the target segments, and ultimately achieving replication of the target sequence.

[0075] The RT template sequences encoding the flap sequences can have different lengths without affecting the efficiency of the AE system. In one embodiment, the length of each RT template sequence is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In one embodiment, the length of each RT template sequence does not exceed 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides. In some embodiments, each RT template sequence is 3-2000, 10-2000, 10-500, 15-500, 15-200, 15-50, or 15-30 nucleotides in length.

[0076] As previously described, the RT template sequences share at least one complementary portion ("paired segment"). In some embodiments, the length of each paired segment is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In one embodiment, the length of each paired segment does not exceed 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides, or 500 nucleotides. In some embodiments, the length of each paired segment is 3-200, 5-200, 10-50, 10-25, or 15-20 nucleotides.

[0077] Alternatively, in addition to the paired segments, each RT template sequence may also include a portion that is not necessarily complementary to each other. In some embodiments, such a non-complementary template sequence is located between the corresponding paired segment and the crRNA / sgRNA or near the PBS sequence.

[0078] In some embodiments, the length of the non-complementary template sequence is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 nucleotides. In some embodiments, the length of the non-complementary template sequence does not exceed 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or 2000 nucleotides. In some embodiments, the length of each non-complementary template sequence is 1-2000, 1-1000, 1-500, 10-500, 15-200, 15-50 or 15-30 nucleotides.

[0079] Alternatively, in addition to the paired fragments, each RT template sequence may or may not include a portion complementary to the genomic DNA near the nicking site of another pegRNA. In some embodiments, each fragment paired with genomic DNA has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 nucleotides. In one embodiment, the length of each paired fragment does not exceed 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150 or 200 nucleotides, or 500 nucleotides.

[0080] The currently disclosed AE technology can amplify target fragments of various lengths. In some embodiments, the length of the target fragment is at least 10, 50, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 100,000, 1,000,000 (1 Mb) or 100,000,000 (100 Mb) or bp, or the entire chromosome.

[0081] The length of the replication is the distance between the two nick sites. Therefore, in some embodiments, the two nicks located on both sides of the target fragment are at least 10, 50, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 100000, 200000, 300000, 1000000, 1000,000 or 100,000,000 or 100,000,000 or 100,000,000 or the entire chromosome. In some embodiments, the two sites are less than 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 200000, 300000, 1000000, 1,000,000 or 100,000,000 or 100,000,000. In some embodiments, they are 2 to 300,000 base pairs apart from each other, more preferably 10 to 50,000 base pairs.

[0082] The pegRNA disclosed in this article contains other elements used in conventional prime editing technology.

[0083] Prime editing is a genome editing technology that can be used to modify the genome of living organisms. Prime editing technology writes new genetic information directly into a target DNA site. The technology uses a fusion protein composed of a catalytically impaired endonuclease (such as Cas9) and an engineered reverse transcriptase (RT), combined with a prime editing guide RNA (pegRNA). The pegRNA is able to recognize the target DNA site and provide new genetic information to replace nucleotides in the target DNA. Prime editing technology enables targeted insertions, deletions, and base transitions without the need for double-strand breaks (DSBs) or donor DNA templates.

[0084] PegRNA can recognize the target nucleotide sequence to be edited and encode new genetic information to replace the target sequence. PegRNA consists of an extended single-stranded guide RNA (sgRNA) or crRNA, which contains a primer binding site (PBS) and a reverse transcriptase (RT) template sequence. During the genome editing process, the 3' end of the cut DNA chain is complementary to the primer binding site of the pegRNA, and the RT template is used as a template to form the edited genetic information and is reverse transcribed into a single-stranded DNA containing the editing information. The sgRNA or crRNA part includes a guide sequence (spacer) and an sgRNA / crRNA backbone structure. The guide sequence can guide the prime editor to the target genomic site.

[0085] In certain embodiments, the pegRNA may further comprise a tail that (a) is capable of forming a hairpin structure or a loop structure with itself, a PBS, an RT template sequence, a crRNA, or a combination thereof, or (b) comprises a poly (A), poly (U), or poly (C) sequence, or an RNA binding domain.

[0086] Cas protein and reverse transcriptase can be provided in the form of fusion protein, and can also be provided separately and combined at the target site (for example, as a complex). In certain embodiments, the fusion protein includes a fusion of a Cas protein (for example, nickase) and a reverse transcriptase. Nickase can be derived from conventional Cas9 proteins, such as SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpf1, LbCpf1, FnCpf1, VQR SpCas9, EQR SpCas9, VRER SpCas9, SpCas9-NG, xSpCas9, RHA FnCas9, KKH SaCas9, NmeCas9, StCas9, CjCas9 or atCas9. An exemplary nickase is Cas9 H840A. The Cas9 enzyme comprises two nuclease domains that can cut double-stranded DNA sequences, wherein the RuvC domain cuts the non-targeting chain and the HNH domain cuts the targeting chain. By introducing the H840A substitution mutation in Cas9, the histidine residue at position 840 is replaced with alanine, thereby inactivating the HNH domain. At this point, only the RuvC domain plays a role, making the catalytically inactivated Cas9 only able to perform single-stranded cleavage, so it is called nickase.

[0087] The traditional PE2 system consists of Cas9 nickase-RT and pegRNA. However, Cas12 protein has not yet been applied in primeediting, mainly due to the lack of corresponding Cas12 nickase. Traditional pegRNA is not expected to function with Cas12. Cas9 nickase introduces single-stranded nicks, while Cas12 protein cuts double strands. Traditional pegRNA includes a single-stranded guide RNA (sgRNA) or only a crRNA, which contains a guide sequence (spacer) and a skeleton (scaffold), as well as a reverse transcriptase (RT) template sequence and a primer binding site (PBS), presenting a spacer-scaffold-RTT-PBS (from 5' to 3') structural configuration. If the target genome is cut into double strands by Cas12 protein, the RTT in pegRNA will not be effectively used as an RT template.

[0088] In the Cas9-based AE system, the RT template is flanked by PBS and crRNA sequences. When the Cas12 protein is used, the RT template and crRNA are located on both sides of the PBS. In some embodiments, the nickase can be a nickase of SpyCas9, SauCas9, NmeCas9, StCas9, FnCas9, CjCas9, AnaCas9 or GeoCas9.

[0089] The Cas protein can be a Cas12 protein, or Cas12a, Cas12b, Cas12f and Cas12i, without any limitation. Examples include AsCpf1, FnCpf1, SsCpf1, PcCpf1, BpCpf1, CmtCpf1, LiCpf1, PmCpf1, Pb3310Cpf1, Pb4417Cpf1, BsCpf1, EeCpf1, BhCas12b, AkCas12b, EbCas12b and LsCas12b.

[0090] The reverse transcriptase is not limited in any way and may include human immunodeficiency virus (HIV) reverse transcriptase, Moloney murine leukemia virus (M-MLV) reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase, and any reverse transcriptase that can function under physiological conditions.

[0091] Amplification editing can be accomplished by transfecting target cells with a pair of pegRNAs or pegRNA / sgRNA and a fusion protein, or with Cas9 nuclease and reverse transcriptase, respectively. Transfection is typically accomplished by introducing a vector into cells. In some embodiments, the editing tool can be introduced directly into the cell in the form of a protein and RNA or a complex thereof. Each molecule can be introduced separately or together, with no limitation on the method.

[0092] The vector can be introduced into the target host cell by known methods, including but not limited to transfection, transduction, cell fusion, electroporation and liposome transfection. The vector may contain various regulatory elements, such as a promoter. In some embodiments, disclosed herein is an expression vector comprising any polynucleotide described herein, for example, an expression vector comprising a polynucleotide encoding a fusion protein and / or a pegRNA.

[0093] In some embodiments, replication occurs in a cell comprising a DNA polymerase. Such replication can occur intracellularly, in vitro, in vitro, or in vivo. For example, the cell can be a prokaryotic cell, a eukaryotic cell, a plant cell, an animal cell, a mammalian cell, or a human cell.

[0094] In certain embodiments, the cell is not in an actively dividing state, i.e., the cell is not undergoing cell division or chromosome replication. In certain embodiments, the cell is further engineered to express a DNA polymerase. Application of AE technology

[0095] DNA fragment amplification has a wide range of applications in clinical and industrial fields.

[0096] For example, duplication / amplification of certain genes may lead to the development of cancer. Therefore, the AE method is able to generate animal disease models (such as cells, mice, fruit flies, etc.) formed by gene duplication and can be used to simulate such pathogenic features, such as repeat expansion diseases and oncogene copy number changes.

[0097] Gene duplication has important applications in plants. The amplification of certain genes or genomic sequences (such as disease resistance genes) has natural advantages in plants to resist diseases, pests and environmental stress. Therefore, AE technology can help plants achieve these advantages. Gene duplication also has application value in microorganisms, for example, to generate gene clusters in fungi, bacteria or yeast.

[0098] Certain diseases in humans (α-globin deficiency) are caused by haploid defects (haploinsufficiency). Therefore, duplication of these missing genes can increase gene expression and restore the normal phenotype of the patient. Chromosomal microdeletions are usually caused by deletions of 0.1Mb ​​to several Mb in one chromosome copy. AE can efficiently amplify genomic regions of 0.1–10Mb. AE can compensate for gene loss due to microdeletions by amplifying the corresponding sequence in the sister chromosome.

[0099] Therapeutic proteins (such as vaccines and antibodies) are already produced on a large scale. The production efficiency may be limited by the number of copies of the coding sequence in the host cell (such as the number of copies in CHO cells), especially the copies integrated into the genome. AE technology can easily and quickly amplify these coding sequences, thereby increasing protein production.

[0100] The cell cycle may be limited by the number of telomere copies or the length of the telomere. In one embodiment, a method is provided for amplifying the telomere or a portion thereof using AE technology. Such amplification may increase the survival rate or replication capacity of the cell, or extend the life span of the organism.

[0101] The present disclosure also provides compositions, kits and packages for such applications. In certain embodiments, a composition, kit or package is provided for replicating a target sequence in the presence of a DNA polymerase, comprising: (a) a Cas protein and a reverse transcriptase, (b) a guide RNA (pegRNA1) for induction editing, including a first CRISPR RNA (crRNA1), a reverse transcriptase template sequence (RT1), (c) another guide RNA (pegRNA) for induction editing, including a second crRNA2 and a second RT2 template sequence, wherein (i) the first RT template sequence includes a first DNA complementary DNA, (ii) the second RT template sequence includes a second complementary DNA, (iii) the first complementary DNA fragment and the second complementary DNA fragment are complementary to each other, (iv) the first pegRNA and the second pegRNA can guide the Cas protein to cut at two sites on both sides of the target fragment, and the cutting occurs on mutually complementary chains.

[0102] This patent also provides another composition, kit or package for replicating a target fragment of a target DNA sequence in the presence of a DNA polymerase, comprising: (a) a Cas protein, (b) a first single-stranded guide RNA (sgRNA1), and (c) a second single-stranded guide RNA (sgRNA2), wherein the first sgRNA and the second sgRNA each have sequences complementary to both sides of the spacer sequence in the target DNA sequence, and there is at least partial complementarity between the two target sites.

[0103] Further, the present disclosure provides a composition, a kit or a package for replicating a target fragment of a target DNA sequence in the presence of a DNA polymerase, comprising: (a) a Cas protein and a reverse transcriptase, (b) a primary editing guide RNA (pegRNA), comprising a first CRISPR RNA (crRNA) and a reverse transcriptase (RT) template sequence, and (c) a single guide RNA (sgRNA), wherein: (i) the RT template sequence comprises a paired fragment, (ii) the pegRNA can guide the Cas protein to cut at a first site near the target fragment of the target DNA sequence, thereby allowing the reverse transcriptase to extend the complementary chain of the target fragment using the RT template sequence as a template to generate a single-stranded DNA sequence, and (iii) the sgRNA can guide the Cas protein to cut at a second site near the target fragment of the target DNA sequence, thereby releasing the complementary chain to form a second single-stranded DNA sequence.

[0104] Each component is further described in the previous sections, which are combined here. example Example 1. Design and testing of expansion editors

[0105] A new system based on primed editing (PE) technology is designed to replicate the desired fragment of the target sequence. This new technology is called "Amplification Editing" (AE). Figure 1 ab is an example of the AE editing process.

[0106] In the example shown, two pegRNA molecules were used. Figure 1 a, each pegRNA contains, in addition to CRISPR RNA (crRNA) / single-stranded guide RNA (sgRNA), a reverse transcriptase (RT) template sequence and a primer binding site (PBS). The PBS can be complementary to the guide sequence (or "spacer sequence") in the crRNA / sgRNA, but is usually a few nucleotides shorter. When the guide sequence binds to the targeted genomic sequence and unwinds the DNA double strand, the PBS can bind to the complementary strand, initiate the reverse transcription process, and use the RT template sequence as a template.

[0107] Unlike the pegRNA of traditional guide editing technology, in each pegRNA in the amplification editing system, the RT template sequence does not need to be homologous to the targeted genomic sequence. In some embodiments, the RT template sequence preferably has a sequence with low or even no homology to the target sequence.

[0108] Instead, the two RT templates share a complementary part. Figure 1As shown in a, in each pegRNA, the RT template includes two parts, one is the RT matching fragment close to the crRNA / sgRNA (or called "complementary part"), and the other is the RT fragment far away from the crRNA / sgRNA, or in Fig.16 As shown in . The two paired (complementary) RT fragments have complementary sequences so that the DNA sequences reverse transcribed from them can pair with each other.

[0109] When the guide sequence binds to the target genomic sequence and unwinds the DNA double strand, the PBS will bind to the complementary strand and initiate the reverse transcription process, using the RT template sequence as a template. Figure 1 As shown in FIG. 1B , the two pegRNAs are designed to cut at the "PAM-out" positions on both sides of the target fragment, and the cutting occurs at two locations of the target fragment (step 110). The RT template is then used as a template for synthesizing single-stranded DNA, thereby introducing two mutually extending single-stranded DNA regions (step 120). The term "PAM-out" herein refers to the fact that the PAM sequence is located outside the region sandwiched by the two target sites (sites recognized by the spacer sequence).

[0110] The two single-stranded DNAs include the reverse transcription product of the RT fragment from the pegRNA and the reverse transcription product from the distal paired (complementary) RT fragment. Due to their complementarity, the two distal fragments can form a double-stranded region by hybridization (step 130). This double-stranded region can then serve as the starting point of the DNA polymerase.

[0111] Using the double-stranded region as a starting point and the single-stranded genomic DNA as a template, a new DNA chain is synthesized and the original genomic DNA is unwound between the two gaps (steps 140-150), ultimately replacing the target sequence (sequence A) with a new fragment that includes the first copy of sequence A, an insertion sequence based on the RT template of two pegRNAs, and the second copy of sequence A. In essence, the amplification editing process (A) copies sequence A and (B) inserts a new sequence based on the RT template between them.

[0112] This case further tested the amplification editing technology in the laboratory. We designed three types of PCR primers to examine the editing results of AE ( Figure 2 a). In-In PCR uses a pair of reverse primers that are inside the amplification region and cannot amplify the unedited sequence. Out-Out PCR uses a pair of primers that target outside the amplification region. In-Out PCR uses one primer that targets outside the amplification region and one primer that targets the insert sequence (generated by the 3' flap).

[0113] We then used two pairs of pegRNAs designed to replicate a 178 bp sequence in the VEGFA locus and a 234 bp sequence in the HEK3 locus in HEK293T cells. PCR bands of the same expected size were detected in AE-edited cells by In-In PCR and In-Out PCR, but not in control cells, indicating that the desired replication was achieved ( Figure 2 b). Two different bands were detected in AE-edited cells using Out-Out PCR, while only one band was detected in control cells, indicating that the target region was amplified ( Figure 2 b) The large band in the Out-Out PCR was analyzed by Sanger sequencing, showing the sequence exactly replicated at the target site with a 20 bp flap sequence inserted in between ( Figure 2 c). Example 2. Characterization and optimization of AE

[0114] To quantify the efficiency of AE, a droplet digital PCR (ddPCR) approach was used. The primer design for the editing type was the same as that for In-InPCR, and the probe was designed to target the sequence of the replication region ( Figure 3 d).

[0115] The length of the paired 3' ssDNA region ranged from 10 bp to 100 bp, and they were complementary to each other. We tested different replication sizes at the VEGFA and C-MYC loci. For replications from ~200 bp to ~8 Kb, the 3' ssDNA length ranged from 30-50 bp, showing high efficiency. In HEK293T cells at the VEGFA locus, the efficiency of 178 bp replication was over 60%, the efficiency of 1 Kb replication was over 40%, and the efficiency of 8 Kb replication was about 20% ( Figure 3 a). The trend of C-MYC locus is similar ( Figure 3 b). Paired 3' ssDNAs can be partially complementary (i.e. "overlapping") at their 3' ends. We used a 30 bp overlap and extended the length of the 3' ssDNA. Our data showed that 30 bp 3' ssDNA showed higher efficiency in ~200 bp replication, while 50 bp 3' ssDNA showed the best efficiency in 8 Kb replication ( Figure 3 c). We also studied the effects of different GC contents in 3' single-stranded DNA and found that 30%-80% GC content of 3' single-stranded DNA can effectively promote replication ( Figure 4 a). To explore the feasibility of simultaneous replication at multiple sites, we co-transfected plasmids encoding two or three pairs of pegRNAs into HEK293T cells. The replication efficiency at each site by multiple editing was comparable to that of single site editing ( Figure 4 b).

[0116] To determine the purity of the editing outcome, we performed deep sequencing of the left and right interfaces of the replicated region as well as the middle interface containing the flap insertion ( Figure 5 a). The purity of the three interfaces of VEGFA-178bp replication and HEK3-234bp replication was close to 100% ( Figure 5 b). The purity of the replication interface from 200 bp to 8 Kb of VEGFA and C-MYC loci was also close to 100%, indicating that the replication of AE was highly accurate ( Figure 5 ce). Example 3. AE is active at different endogenous sites in multiple cell lines

[0117] AE has been edited in the three endogenous sites mentioned above. We will further explore the replication ability of AE in other endogenous sites, including AAVS1, RUNX1, and HEK4, and quantify the replication efficiency by ddPCR. We found that the replication rate of ~200bp ranged from 56.3% to 68.5%, the replication rate of ~1-2Kb ranged from 28.5% to 47.8%, and the replication rate of ~7-9Kb ranged from 20.4% to 33.3%, all of which were found in the editing of AE in HEK293T cells ( Figure 6 a).

[0118] Next, we examined whether AE was active in other cell lines, such as human Huh-7 cells, human K562 cells, human U2OS cells, and mouse N2a cells. By ddPCR, we found that the replication efficiency of AE in Huh-7 cells was 1.7% to 34.6%, in K562 cells was 18.9% to 66.1%, in U2OS cells was 4.9% to 25.2%, and in N2a cells was 20.0% to 85.5% ( Figure 6 We verified replication events in these cells by In-In and In-Out PCR ( Figure 7 ac). Example 4. AE generates tandem duplication events

[0119] Next, we explored whether AE could replicate DNA fragments smaller than 150 bp. To this end, we designed AE to replicate DNA fragments ranging from 20-130 bp, with editing efficiencies ranging from 28.6% to 52.3% ( Figure 8 ab). It should be noted that when the replication fragment size is 20 bp, the spacer sequences of the two pegRNAs are partially complementary, and the primer binding site (PBS) overlaps with the spacer sequence of another pegRNA ( Figure 8a). Therefore, each 3' single-stranded DNA generation process may be sequential. We amplified the editing products of small fragments by Out-Out PCR and performed deep sequencing analysis. The results showed that the replicated sequence was consistent with the expected ( Figure 8 c). As expected, we also found a duplication fragment with a small deletion, which may be due to the two cuts being too close to each other, making it difficult for the 20bp to be replicated smoothly. More interestingly, we found three groups of tandem repeats consisting of a 20bp sequence and an intervening sequence, indicating that duplication may occur multiple times ( Figure 8 ac, Fig. 9 a).

[0120] We then used Out-Out PCR to amplify the 234 bp duplication region of the HEK3 locus in single-cell clones. Gel electrophoresis analysis showed that the number of tandem repeats ranged from two to nine, and the sequences of these repeats were verified by Sanger sequencing ( Fig. 9 bc). Although more tandem repeats may occur in theory, their length may exceed the detection range of genomic PCR. After each round of replication, the recognition site of each pegRNA remains intact, thus supporting the occurrence of the next round of replication. When the mechanism of the next round of replication is consistent with the first round of replication, 2 n Multiple repeat sequences (e.g. 2A, 4A, 8A) Fig. 9 a Left figure). In addition, the 3' single-stranded DNA may also pair with the inserted fragment generated in the previous round of replication, thereby generating different numbers of repeats ( Fig. 9 a (right figure).

[0121] Consistent with the above observations, we detected over 100% duplication efficiency (relative to the reference gene) at the VEGFA and RUNX1 loci in K562 cells by ddPCR analysis ( Fig.10 a). To further evaluate the efficiency of AE serial replication, we used restriction endonucleases to fragment the generated tandem repeats. In the fragmented samples, the serially replicated fragments will be distributed to different ddPCR droplets, thereby improving the replication efficiency ( Fig.10 b). In contrast, samples with only one replication event showed no significant difference in results regardless of whether they were fragmented or not. In K562 cells, the replication efficiency of VEGFA-178bp reached 808.7%, while the replication efficiency of VEGFA-1Kb increased from 66.1% to 199.0%. After fragmentation, the replication efficiency was significantly higher than that of the unfragmented samples ( Fig.10 c). A similar trend was observed in the HEK3 locus of K562 cells ( Fig.10d). In HEK293T cells, the replication of fragments of approximately 200 bp and 1 Kb also showed a consistent trend ( Figure 3 fg). When the size of the replication fragment reached 8Kb, no significant difference was observed before and after fragmentation in K562 cells, while in HEK293T cells, the replication efficiency was slightly improved after fragmentation ( Fig.10 ef). These data indicate that the frequency of serial replication decreases with increasing replication fragment size. Example 5. Functional assay and potential applications of AE

[0122] To demonstrate that AE can restore gene expression through duplication, we established a stable cell line in which a small deletion (53 bp) of the GFP sequence was present. A pair of pegRNAs with a PAM-out design was used to duplicate the GFP region and insert the small deletion fragment to restore GFP expression ( Fig.11 a). AE successfully generated ~30% GFP-positive cells through gene duplication ( Fig.11 b).

[0123] α-thalassemia is a common blood disorder usually caused by mutations in the HBA1 and HBA2 genes (two almost identical genes). The most common form is a 3.7Kb deletion (-α3.7) of the HBA1 and HBA2 genes, resulting in the production of a fusion HBA gene that is identical to the HBA1 gene. We constructed the -α3.7 genotype in HEK293T cells using CRISPR technology and replicated the fusion HBA gene using AE technology, thereby repairing the -α3.7 deletion ( Fig.11 c). The replication efficiency of the HBA gene reached 16.4% ( Fig.11 d).

[0124] To verify whether AE treatment can cause functional changes in endogenous genes, we applied AE to amplify the stem-loop region of microRNA-21 (miR-21) Fig.12 a). In HEK293T and K562 cells, the replication efficiency of the miR-21 stem-loop region was 94.6% and 189.0%, respectively, indicating that the region underwent multiple rounds of amplification ( Fig.12 b). Compared with control cells, the expression levels of miR-21 in HEK293T and K562 cells were significantly increased by 5.9-fold and 6.2-fold, respectively, after 7 days of AE treatment ( Fig.12 c). At the same time, the expression levels of miR-21 target genes decreased, indicating that the replication process was functionally effective ( Fig.12 d). Example 6. AE can copy 30Kb to 100Mb

[0125] We explored whether AE is capable of replicating large genomic regions ranging from 30 Kb to 100 Mb. By designing multiple pairs of pegRNAs with intervals of 30, 60, and 100 Kb, respectively, and applying them to chromosome 6 (Chr 6) or chromosome 9 (Chr 9), we used In-In PCR to detect the presence of replication events. To obtain the longest possible amplified sequence, we performed In-In PCR experiments using multiple pairs of primers. The control samples failed to show any bands in the In-In PCR, while the AE-treated samples showed the expected PCR products with sizes ranging from 3.6 to 5.0 Kb ( Fig.13 a). These PCR products were sequenced by Sanger sequencing with multiple primers, and the results showed that the replicated sequences were correct ( Fig.13 a). The replication efficiency determined by ddPCR ranged from 7.5% to 31.7% for 30-60Kb replication and 28.8% for 100Kb replication ( Fig.13 c).

[0126] Inspired by the above results, we further explored the possibility of AE replicating the genome at the Mb level. We first tested the efficiency of replicating 1Mb and 3Mb on chromosomes 6, 9, and 12. The replication efficiency of the 1Mb region ranged from 9.0% to 27.7%, and the replication efficiency of the 3Mb region ranged from 2.7% to 7.6% ( Fig.13 d). To confirm the presence of Mb-level duplication, we measured the copy number of three different genes within the duplicated region of chromosome 12. In control cells, the copy number of these genes was approximately 3, indicating that this region was triploid. In AE-treated monoclonal cells, the copy number of these genes was approximately 4, indicating that duplication occurred in this region of chromosome 12 ( Fig.13 b).

[0127] We then explored the feasibility of replicating genomic regions ranging from 10 to 100 Mb. The efficiency of chromosome-scale replication ranged from 0.55% to 2.5% for chromosomes 6 and 9 ( Fig.13 f). It is noteworthy that the size of chromosome 6 is 172Mb, and AE successfully replicated a 100Mb region with an efficiency of 1.1% ( Fig.13 f).

[0128] To further confirm the occurrence of large-scale genomic duplication, we used fluorescent in situ hybridization (FISH) technology, using DNA probes targeting the duplicated region of the genome. The STAT6 gene is located in the 3Mb duplicated region of chromosome 12. We used a previously validated DNA probe targeting the STAT6 gene to visualize this duplicated region and used a DNA probe targeting the centromere region as a control ( Fig.14 a). In wild-type cells, two red dots surround one green dot, while monoclonal cells treated with AE show four red dots surrounding one green dot, indicating that the STAT6 region has been duplicated ( Fig.14 a). On chromosome 6, a 100 Mb duplication region occupies most of its long arm (110 Mb). We used a previously validated DNA probe targeting the ESR1 gene to localize to this duplication region ( Fig.14 b). AE edited cells have a significantly extended long arm of chromosome 6 (from 110Mb in the control group to 210Mb in the edited group), and the duplication region within this region has been confirmed ( Fig.14 b) Together, these data confirm the existence of AE chromosome arm duplications in cells ranging from 1-100 Mb. Example 7. Various PAM-out methods for DNA replication

[0129] In this study, we explored whether DNA replication can be achieved by methods other than paired pegRNA. When the Cas9 / sgRNA complex targets DNA, the spacer sequence of the sgRNA binds to its complementary sequence, resulting in a free non-targeted DNA strand and a small 3' single-stranded DNA. We first investigated the replication efficiency of 3' single-stranded DNA with complementary sequences of 10 bp or less. The results showed that the replication efficiencies of 3' single-stranded DNA with 3 bp and 8 bp complementary sequences were 8.5% and 12.6%, respectively ( Fig.15 a). Interestingly, in the sample without any complementary 3' ssDNA generated by RTT of pegRNA, the replication efficiency was 1.9%, and this low replication frequency may be due to the presence of minor homology in the sgRNA binding site. Based on this observation, we believe that pegRNA / sgRNA or sgRNA / sgRNA combinations can be designed with shared complementary sequences in the PAM-out direction to enable annealing of the 3' ssDNA ( Fig.16 a) In addition, it is also feasible to design a 3' single-stranded single to be partially complementary to the genomic sequence near the nick of another pegRNA, and to be partially complementary to each other.

[0130] We further studied the effect of pegRNA / sgRNA combinations with complementary sequences in DNA replication. Specifically, we designed the pegRNA RTT to have 8bp or 0bp complementary sequences between the sgRNA targeting site. The experimental results showed that the replication efficiency of paired pegRNAs at the RUNX1, VEGFA, and AAVS1 gene loci was 32.8%-71.0%, while the replication efficiency of the pegRNA / sgRNA combination with 8bp complementary sequences was between 3.9% and 24.3% ( Fig.16b). Although the efficiency of the pegRNA / sgRNA combinations with 0 bp complementary sequences was lower than that of the combinations designed with 8 bp complementary sequences, they still showed significant editing effects, probably due to the presence of 3-4 bp microhomology between the RTT and the sgRNA targeting site ( Fig.16 b). We analyzed the replication interface of the pegRNA / sgRNA combination by deep sequencing, and the results showed that the purity of the combined samples with 8bp complementary sequence ranged from 53.9% to 87.9%, while the purity of the 0bp complementary sequence samples ranged from 0% to 9.5% ( Fig.16 c) These results indicate that precise complementary sequence design is key to maintaining efficient and precise editing of AE.

[0131] Next, we tried a pair of sgRNAs and designed 4–8 bp complementary sequences in the direction of PAM-out, targeting the genomic sequence near the cut ( Fig.16 a). Through In-In PCR analysis, we found that bands were clearly observed in samples treated with sgRNAs and Cas9 nuclease, while there were no bands in the control group ( Fig.16 d). The second generation data at the editing group interface showed that the purity of the 8bp complementary sequence was as high as 96.6%, indicating that high-precision DNA replication can still be achieved even without the participation of nCas9-RT and pegRNA ( Fig.16 e). ***

[0132] The disclosure scope of this patent is not limited to the specific embodiments described, and these examples are only single instances of this patent, and any functionally equivalent composition or method belongs to the scope of this patent. For those skilled in the art, various modifications and changes can be made to the methods and compositions in this disclosure without departing from the spirit and scope of this disclosure. Therefore, modifications and changes to the contents described herein, as long as they comply with the scope of the attached claims and their equivalents, are within the scope of protection of this patent.

[0133] All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A method for replicating a target fragment in a target DNA sequence in the presence of a DNA polymerase, comprising contacting the target DNA sequence with the following components: (a) a Cas protein and a reverse transcriptase; (b) a first PE guide RNA (Prime editing guide RNA, pegRNA), the pegRNA comprising a first CRISPR RNA (crRNA) and a first reverse transcriptase (RT) template sequence; (c) a second pegRNA, the pegRNA comprising a second crRNA and a second RT template sequence; wherein: (i) the first RT template sequence includes a first pairing fragment; (ii) the second RT template sequence includes a second pairing fragment; (iii) the first pairing fragment is complementary to the second pairing fragment; (iv) the first pegRNA and the second pegRNA guide the Cas protein to cut the two strands of the target DNA at two positions on both sides of the target fragment in the target DNA sequence, This allows: (1) the reverse transcriptase to extend the two complementary strands of the target fragment using the first and second RT template sequences as templates, generating two single-stranded DNA sequences; (2) the two single-stranded flap sequences form a double-stranded region, allowing the DNA polymerase to extend the double-stranded region to replicate each strand of the target fragment, Thereby, the target fragment is replicated, and an insert fragment is inserted between the two replicated target fragments, wherein one strand of the insert fragment includes the reverse complementary sequence of the first fragment, the first mate fragment and the second fragment.

2. According to the method of claim 1, wherein the first pegRNA further includes a first primer binding site (PBS) and a first spacer sequence, and the second pegRNA further includes a second PBS and a second spacer sequence, so that the pegRNA can guide the Cas protein to two positions on both sides of the target fragment and initiate reverse transcription.

3. The method according to claim 1 or 2, wherein the length of the first and second RT template sequences is 0 to 2000 nucleotides, preferably 15 to 500 nucleotides.

4. The method according to any of the preceding claims, wherein the length of the first and second paired fragments is 0 to 1000 nucleotides, preferably 3 to 200 nucleotides or 3 to 50 nucleotides, more preferably 30-100 nucleotides.

5. A method according to any one of claims 2 to 4, wherein the first and second RT template sequences each further include a non-complementary template sequence that is not complementary to the other sequence, wherein each non-complementary template sequence is located between the corresponding paired fragment and the crRNA, or between the corresponding paired fragment and the PBS.

6. The method according to claim 5, wherein the length of each non-complementary template sequence is 1 to 2000 nucleotides, preferably 1 to 1000 nucleotides or 1 to 500 nucleotides.

7. The method according to any of the preceding items, wherein the two sites on both sides of the target fragment are 2 to 1 billion base pairs apart, preferably 10 to 5 million base pairs apart.

8. The method according to any of the preceding claims, wherein each RT template sequence further comprises an additional sequence immediately adjacent to the paired fragment, wherein the two additional sequences are complementary to the target DNA sequence and are at least partially complementary to each other.

9. A method for replicating a target fragment in a target DNA sequence in the presence of a DNA polymerase, comprising contacting the target DNA sequence with the following components: (a) a Cas protein; (b) a first sgRNA or a tracrRNA; (c) a second sgRNA or tracrRNA, wherein the first sgRNA or tracrRNA and the second sgRNA or tracrRNA each have sequence complementarity with target sites on both sides of the target fragment in the target DNA sequence, and the two target sites have at least partial complementarity; wherein: The first sgRNA or tracrRNA, in the presence of the Cas protein, binds to one chain and cuts the complementary chain of the target site, releasing the complementary chain as the first single-stranded DNA; the second sgRNA or tracrRNA, in the presence of the Cas protein, binds to one chain and cuts the complementary chain of the target site, releasing the opposing chain as the second single-stranded DNA; the first single-stranded DNA combines with the second single-stranded DNA to form a double-stranded region, allowing the DNA polymerase to extend the double-stranded region to replicate the target sequence, thereby replicating the sequence between the two target sites.

10. The method according to item 9, wherein the partial complementarity comprises complete complementarity of at least 3, 4, 5, 6, 7 or 8 consecutive nucleotides.

11. A method for replicating a target fragment in a target DNA sequence in the presence of a DNA polymerase, comprising contacting the target DNA sequence with the following components: (a) a Cas protein and a reverse transcriptase; (b) a pegRNA comprising a first CRISPR RNA (crRNA) and a reverse transcriptase (RT) template sequence; (c) an sgRNA or a tracrRNA; wherein: (i) The RT template sequence includes a paired fragment; (ii) pegRNA guides the Cas protein to cut at the first position on the target DNA sequence close to the target fragment, thereby allowing the reverse transcriptase to extend the opposite strand of the target fragment using the RT template sequence as a template to generate a single-stranded DNA sequence; (iii) sgRNA or tracrRNA guides the Cas protein to cut at the second position on the target DNA sequence close to the target fragment, thereby releasing the opposite strand as a second single-stranded DNA sequence; thereby, the two single-stranded DNA sequences form a double-stranded region, allowing the DNA polymerase to extend the double-stranded region to replicate the target fragment, thereby replicating the target fragment.

12. A method according to any preceding claim, wherein the target DNA sequence is located in a cell, which cell can be selected from a eukaryotic cell or a prokaryotic cell, a plant cell, an animal cell, a mammalian cell or a human cell.

13. The method according to claim 12, wherein the cells are dividing cells.

14. The method according to claim 12, wherein the cells are not dividing cells.

15. A method according to any preceding claim, wherein the target fragment is a telomere or a fragment thereof.

16. A method according to any preceding claim, performed in vitro.

17. The method according to any one of items 1 to 15, wherein the method is performed in vivo.

18. A method according to any one of items 1 to 17, wherein the Cas protein is a nickase.

19. The method according to claim 18, wherein each pegRNA comprises a first or second crRNA, a first or second pairing fragment, a first or second fragment, and a first or second PBS (from 5' to 3' direction).

20. The method according to item 18 or 19, wherein the cutting enzyme is a Cas9 protein containing an inactive HNH domain, which cuts the target chain.

21. The method according to item 20, wherein the cleavage enzyme is SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpf1, LbCpf1, FnCpf1, VQR SpCas9, EQR SpCas9, VRER SpCas9, SpCas9-NG, xSpCas9, RHA FnCas9, KKH SaCas9, Cutting enzymes of NmeCas9, StCas9, CjCas9 or atCas9.

22. A method according to any one of items 1 to 19, wherein the Cas protein is a Cas12 protein.

23. The method according to claim 22, wherein each pegRNA comprises a first or second crRNA, a first or second PBS, a first or second fragment and a first or second paired fragment (from 3' to 5' direction).

24. The method of item 22 or item 23, wherein the Cas12 protein is Cas12a, Cas12b, Cas12f or Cas12i.

25. The method according to item 24, wherein the Cas12 protein is selected from the following proteins: AsCpf1, FnCpf1, SsCpf1, PcCpf1, BpCpf1, CmtCpf1, LiCpf1, PmCpf1, Pb3310Cpf1, Pb4417Cpf1, BsCpf1, EeCpf1, BhCas12b, AkCas12b, EbCas12b and LsCas12b.

26. A method according to any one of claims 2-25, wherein the first pegRNA or the second pegRNA further comprises a tail that (a) is capable of forming a hairpin structure or loop with itself, PBS, RT template sequence, crRNA or a combination thereof, or (b) comprises a poly(A), poly(U) or poly(C) sequence, or an RNA binding domain.

27. A method according to any preceding claim, wherein the reverse transcriptase is M-MLV reverse transcriptase or a reverse transcriptase capable of functioning under physiological conditions.

28. A method according to any one of claims 1 to 5, wherein the Cas protein and reverse transcriptase are provided as nucleotide sequences encoding the respective proteins, or are provided in the form of proteins.

29. A method according to any preceding claim, wherein each pegRNA is provided as a recombinant DNA or RNA molecule encoding the pegRNA.

30. A method according to any preceding claim, wherein the replicated target fragment and the inserted fragment are further replicated.

31. A composition, kit or package for replicating a target fragment in a target DNA sequence in the presence of a DNA polymerase, comprising: (a) Cas protein and reverse transcriptase; (b) a first pegRNA, which includes a first CRISPR RNA (crRNA) and a first reverse transcriptase (RT) template sequence; (c) a second pegRNA, which includes a second crRNA and a second RT template sequence; wherein: (i) the first RT template sequence includes a first pairing fragment; (ii) the second RT template sequence includes a second pairing fragment; (iii) the first pairing fragment is complementary to the second pairing fragment; (iv) the first pegRNA and the second pegRNA can guide the Cas protein to cut the two chains of the target DNA at two positions on both sides of the target fragment in the target DNA sequence.

32. A composition, kit or package for replicating a target fragment in a target DNA sequence in the presence of a DNA polymerase, comprising: (a) Cas protein; (b) a first sgRNA or tracrRNA; (c) a second sgRNA or tracrRNA, wherein the first sgRNA or tracrRNA and the second sgRNA or tracrRNA each have sequence complementarity with target sites on both sides of the target fragment in the target DNA sequence, and the two target sites have at least partial complementarity.

33. A composition, kit or package for replicating a target fragment in a target DNA sequence in the presence of a DNA polymerase, comprising: (a) Cas protein and reverse transcriptase; (b) a pegRNA, the pegRNA comprising a first CRISPR RNA (crRNA) and a reverse transcriptase (RT) template sequence; (c) an sgRNA or tracrRNA; wherein: (i) the RT template sequence comprises a paired fragment; (ii) pegRNA can guide the Cas protein to cut at the first position on the target DNA sequence, close to the target fragment, thereby allowing the reverse transcriptase to extend the opposite strand of the target fragment using the RT template sequence as a template to generate a single-stranded DNA sequence; (iii) sgRNA or tracrRNA can guide the Cas protein to cut at the second position on the target DNA sequence, close to the target fragment, thereby releasing the opposite strand as a second single-stranded DNA sequence.

Images