CN113564164A - Carrier and method for improving pilot editing efficiency - Google Patents

Abstract

The invention discloses a carrier and a method for improving the efficiency of pilot editing. The vector capable of improving the lead editing efficiency can transcribe the pegRNA, the pegRNA is an RNA molecule obtained by connecting a sgRNA targeting a target DNA fragment, a reverse transcription template and a primer binding site, and the 3' end of the primer binding site and/or the middle of the primer binding site are not matched with nucleotides of a spacer of the sgRNA. Experiments prove that the method can effectively improve the editing efficiency of the lead editing system in the plant by introducing the mutant base in the middle position of the PBS or introducing the mutant base at the 3' end of the PBS, and the DNA lead editing system and the related vector obtained by the method have good application prospects.

Description

Carrier and method for improving pilot editing efficiency

Technical Field

The invention relates to a carrier and a method for improving the efficiency of pilot editing in the field of biotechnology.

Background

Precise gene editing plays an important role in crop breeding and genome function research. However, the editing window of the single base editor (ABE and CBE) is narrow, the traditional homologous recombination repair (HDR) needs exogenous DNA template, the system is complex and the efficiency is low, and the development of related work is greatly limited. The new tool for precise gene editing, pe (prime editors), emerged since 10 months 2019, brought a major breakthrough in the field of gene editing (Anzalone, Randolph et al). The new tool PE is based on a CRISPR-Cas9 system, a reverse transcription template (rtT) containing new genetic information and a Primer Binding Site (PBS) sequence are added at the 3' end of a single-stranded guide RNA (sgRNA) in a lead editing guide RNA, Cas9 nickase (H840A) and reverse transcriptase (M-MLV) are fused to form a new fusion protein, and the new genetic information carried in the reverse transcription template can be ' written ' into a plant genome under the action of the reverse transcriptase.

In animal cells, the PE system can effectively realize free conversion of 12 single bases and precise insertion and deletion of multiple bases without introducing Double Strand Breaks (DSBs) and without donor DNA templates. In 2020, several groups of scientific research teams have published articles successively to describe the application of the DNA leader editing system in plants, but the editing efficiency needs to be further improved (Lin, Zong et al.2020). Therefore, it is necessary to develop a method for significantly improving the efficiency of the pilot editing to improve the usability and friendliness of the system and make the precise editing play a greater role.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the accurate editing efficiency of the PE system.

In order to solve the above technical problems, the present invention provides a DNA leader editing system, which comprises a pegRNA that is an RNA molecule obtained by linking a sgRNA targeting a DNA fragment of interest, a reverse transcription template, and a Primer Binding Site (PBS), wherein the 3' end of the primer binding site and/or the middle thereof is mismatched with the nucleotide of the spacer of the sgRNA.

In the above system, one, two or three of the three nucleotides at the 3' end of the primer binding site do not match the corresponding nucleotides of the spacer of the sgRNA; and/or the seventh nucleotide from the 3' end of the primer binding site or other sequences thereof do not match with the corresponding nucleotide of the spacer of the sgRNA.

The other sequence refers to other nucleotides of PBS except for the three nucleotides at the 3 'end and the seventh nucleotide from the 3' end.

In the system, the DNA leader editing system can also contain a fusion protein formed by fusing Cas9 nickase (H840A) and reverse transcriptase M-MLV.

The invention also provides a vector containing a DNA molecule for transcribing the pegRNA.

The sequence of the DNA molecule can be 515-639 th site of the sequence 4.

The vector may contain greater than 1 DNA molecule containing the pegRNA transcribed.

In the vector, the transcription of the DNA molecule containing the pegRNA can be driven by the DNA molecule shown in the 1 st-994 th position of the sequence 3 in the sequence table or the DNA molecule shown in the 1 st-437 th position of the sequence 4 in the sequence table and/or the DNA molecule shown in the 811 th-1334 th position of the sequence 4 in the sequence table.

The vector may further contain a gene encoding the fusion protein.

The encoding gene of the fusion protein can be 2006-8359 th position of the sequence 1.

The carrier can also contain tRNA gene and RNA ribozyme gene, and the tRNA gene, the pegRNA gene and the RNA ribozyme gene are optionally connected in sequence in the same gene expression frame.

The vector may also contain a resistance marker gene.

The application of the system or the vector in gene editing also belongs to the protection scope of the invention.

The invention also provides a gene editing method, which comprises the step of introducing the vector into an object to be edited to realize gene editing.

The body to be edited may be a plant, such as rice.

The application of the system or the vector or the gene editing method in plant breeding also belongs to the protection scope of the invention.

Experiments prove that the method can effectively improve the editing efficiency of the pilot editing system in the plant by introducing the mutant base in the middle position of the PBS or introducing the mutant base at the 3' end of the PBS, and the pilot editing system obtained by the method has good application prospect.

Drawings

FIG. 1 shows the schematic structure of the vector pG3H-PE2-35C containing the pegRNA and the vector pG3H-PE2-U3 and further the construction process of the vector.

FIG. 2 is a sequence analysis. The upper panel shows the sequence, length and target sequence of rtT & PBS when a mutation is introduced at position I1879; the OsACC gene is a rice acetyl coenzyme A carboxylase gene; target refers to the Target of the pegRNA, rtT refers to the reverse transcription template, PBS guide binding site. Part 2 is the rtT & PBS sequence with different mutated nucleotides introduced into PBS.

Figure 3 is the efficiency of exact editing in protoplasts of different vectors based on the mutant PBS strategy.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

The pegRNA is formed by adding a reverse transcription template (rtT) containing new genetic information and a Primer Binding Site (PBS) sequence at the 3' end of a single-stranded guide RNA (sgRNA), and the pegRNA is designed according to a target site needing editing. In the examples described below, the pegR1 and the pegR2 are identical. Spacer and sgRNA support constitute complete sgRNA.

Table 1 below is the primers, leader editing guide rna (pegrna) related sequences used in the examples:

TABLE 1 primer, leader editing guide RNA (pegRNA) related sequences

Example 1 construction of leader editing binary vectors

Construction of vector for driving pegRNA expression by U3 promoter

1. Primers mAmp-BsF/tMet-BsF (i.e.primer pair consisting of mAmp-BsF and tMet-BsF in Table 1) and HDV-BsR2/mAmp-BsR (i.e.primer pair consisting of HDV-BsR2 and mAmp-BsR in Table 1) were used, respectively, the cloning vector is formed by reversely inserting a 1879RP0 fragment shown as a sequence 5 in a sequence table by a synthetic vector pUC57-1879RP0(pUC57-1879RP0 is a cloning vector formed by inserting a reverse transcription template and a primer binding site sequence which are contained in the fragment into lacZ of pUC57-simple, and is shown as 1879RP0-rtT/PBS in a table 1) as a template, and PCR is carried out by using a primer HDV-BsF2/tMet-BSR (a primer pair consisting of HDV-BsF2 and tMet-BSR in the table 1) and a synthetic fragment OsWS2 (the sequence of which is shown as a sequence 2 in the sequence table) as a template. Mixing and recovering the obtained three PCR products, performing enzyme digestion by BsaI, purifying and recovering the enzyme digestion product again to obtain a purified enzyme digestion product, performing self-connection on the obtained purified enzyme digestion product, and naming the obtained recombinant vector with a correct sequence as a recombinant vectorpUC57-1879RP02. The pegRNA-related structure of pUC57-1879RP02 is as follows: BsaI-pegR1-HDV-OsU3t-TaU3p-tMet-pegR 2-BsaI.

Primers mAmp-BsF/tMet-BsF (i.e., the primer pair consisting of mAmp-BsF and tMet-BsF in Table 1) and HDV-BsR2/mAmp-BsR (i.e., the primer pair consisting of HDV-BsR2 and mAmp-BsR in Table 1) were used, respectively, to synthesizeThe vector pUC57-1879RP1b (pUC57-1879RP1b is a cloning vector formed by reversely inserting a 1879RP1b fragment shown as a sequence 6 in a sequence table into lacZ of pUC57-simple, and reverse transcription templates and primer binding site sequences contained in the fragment are shown as 1879RP1b-rtT/PBS in Table 1) are used as templates for PCR amplification, and primers HDV-BsF2/tMet-BSR (a primer pair formed by HDV-BsF2 and tMet-BSR in Table 1) are used as templates for PCR amplification to synthesize a fragment OsWS2 (the sequence of which is shown as a sequence 2 in the sequence table). Mixing and recovering the obtained three PCR products, performing enzyme digestion by BsaI, purifying and recovering the enzyme digestion product again to obtain a purified enzyme digestion product, performing self-connection on the obtained purified enzyme digestion product, and naming the obtained recombinant vector with a correct sequence as a recombinant vectorpUC57-1879-RP1b2。pUC57-1879-RP1b2Identical to the pegRNA-related structure of pUC57-1879RP02, and different in PBS sequence.

PCR was carried out using primers mAmp-BsF/tMet-BsF (i.e., a primer pair consisting of mAmp-BsF and tMet-BsF in Table 1) and HDV-BsR2/mAmp-BsR (i.e., a primer pair consisting of HDV-BsR2 and mAmp-BsR in Table 1), respectively, as a cloning vector consisting of lacZ in which pUC57-1879RP2b (pUC57-1879RP2b is 1879RP2b fragment shown as sequence 7 in the sequence Listing was inserted in reverse into pUC57-simple, as a template, a reverse transcription template and a primer binding site sequence contained in this fragment are as represented by 1879RP2b-rtT/PBS in Table 1), and as a primer pair consisting of HDV-BsF2/tMet-BsR (HDV-BsF 2 and tMet-BsR in Table 1), as a template, as a synthetic fragment 2 (whose sequence is shown by sequence Listing in the sequence Listing). Mixing and recovering the obtained three PCR products, performing enzyme digestion by BsaI, purifying and recovering the enzyme digestion product again to obtain a purified enzyme digestion product, performing self-connection on the obtained purified enzyme digestion product, and naming the obtained recombinant vector with a correct sequence as a recombinant vectorpUC57-1879-RP2b2。pUC57-1879-RP2b2AndpUC57-1879RP02the related structures of the pegRNA are the same, and the PBS sequences are different.

The primers mAmp-BsF/tMet-BsF (i.e., the primer pair consisting of mAmp-BsF and tMet-BsF in Table 1) and HDV-BSR2/mAmp-BSR (i.e., the primer pair consisting of HDV-BSR2 and mAmp-BSR in Table 1) were used to synthesize the vector pUC57-1879RP3b (pUC57-1879RP3b as the reverse insertion of 1879RP3b fragment shown in sequence 8 in the sequence ListingA cloning vector consisting of pUC57-simple lacZ, wherein the fragment contains reverse transcription template and primer binding site sequence shown as 1879RP3b-rtT/PBS in Table 1) as template for PCR amplification, and primer HDV-BsF2/tMet-BSR (primer pair consisting of HDV-BsF2 and tMet-BSR in Table 1) is used for PCR amplification, and synthetic fragment OsWS2 (the sequence shown as sequence 2 in the sequence table) is used as template for PCR amplification. Mixing and recovering the obtained three PCR products, performing enzyme digestion by BsaI, purifying and recovering the enzyme digestion product again to obtain a purified enzyme digestion product, performing self-connection on the obtained purified enzyme digestion product, and naming the obtained recombinant vector with a correct sequence as a recombinant vectorpUC57-1879-RP3b2。pUC57-1879-RP3b2AndpUC57-1879RP02the related structures of the pegRNA are the same, and the PBS sequences are different.

PCR was carried out using primers mAmp-BsF/tMet-BsF (i.e., a primer pair consisting of mAmp-BsF and tMet-BsF in Table 1) and HDV-BsR2/mAmp-BsR (i.e., a primer pair consisting of HDV-BsR2 and mAmp-BsR in Table 1), respectively, as a cloning vector consisting of lacZ in which pUC57-1879RP4b (pUC57-1879RP4b is 1879RP4b fragment shown as sequence 9 in the sequence Listing) was inserted in reverse into pUC57-simple, as a template, a reverse transcription template and a primer binding site sequence included in this fragment are 1879RP4b-rtT/PBS in Table 1), and as a primer pair consisting of HDV-BsF2/tMet-BsR (HDV-BsF 2 and tMet-BsR in Table 1), as a template, as a synthetic fragment 2 (whose sequence is shown as sequence Listing). Mixing and recovering the obtained three PCR products, performing enzyme digestion by BsaI, purifying and recovering the enzyme digestion product again to obtain a purified enzyme digestion product, performing self-connection on the obtained purified enzyme digestion product, and naming the obtained recombinant vector with a correct sequence as a recombinant vectorpUC57-1879-RP4b2。pUC57-1879-RP4b2AndpUC57-1879RP02the related structures of the pegRNA are the same, and the PBS sequences are different.

Primers mAmp-BsF/tMet-BsF (i.e., the primer pair consisting of mAmp-BsF and tMet-BsF in Table 1) and HDV-BSR2/mAmp-BSR (i.e., the primer pair consisting of HDV-BSR2 and mAmp-BSR in Table 1) were used, respectively, to synthesize a vector pUC57-1879RP5b (pUC57-1879RP5b is a cloning vector consisting of a 1879RP5b fragment shown as sequence 10 in the sequence table and inserted in reverse direction into lacZ of pUC57-simple, and a reverse transcription template and a primer combination contained in the fragment are usedThe site sequence is represented by 1879RP5b-rtT/PBS in the table 1) as a template, and the primer HDV-BsF2/tMet-BSR (a primer pair consisting of HDV-BsF2 and tMet-BSR in the table 1) is utilized to carry out PCR amplification by taking the synthetic segment OsWS2 (the sequence of which is represented by the sequence 2 in the sequence table) as a template. Mixing and recovering the obtained three PCR products, performing enzyme digestion by BsaI, purifying and recovering the enzyme digestion product again to obtain a purified enzyme digestion product, performing self-connection on the obtained purified enzyme digestion product, and naming the obtained recombinant vector with a correct sequence as a recombinant vectorpUC57-1879-RP5b2。pUC57-1879-RP5b2AndpUC57-1879RP02the related structures of the pegRNA are the same, and the PBS sequences are different.

PCR was carried out using primers mAmp-BsF/tMet-BsF (i.e., a primer pair consisting of mAmp-BsF and tMet-BsF in Table 1) and primer HDV-BSR2/mAmp-BSR (i.e., a primer pair consisting of HDV-BSR2 and mAmp-BSR in Table 1), respectively, using a cloning vector consisting of pUC57-1879RP6b (pUC57-1879RP6b is 1879RP6b fragment shown as sequence 11 in the sequence Listing and inserted in reverse into lacZ of pUC57-simple, a reverse transcription template and a primer binding site sequence included in the fragment are shown as 1879RP6b-rtT/PBS in Table 1), and using primers HDV-BsF2/tMet-BSR (a primer pair consisting of HDV-BsF2 and tMet-BSR (sequence No. 2 (whose sequence is shown as sequence Listing). Mixing and recovering the obtained three PCR products, performing enzyme digestion by BsaI, purifying and recovering the enzyme digestion product again to obtain a purified enzyme digestion product, performing self-connection on the obtained purified enzyme digestion product, and naming the obtained recombinant vector with a correct sequence as a recombinant vectorpUC57-1879-RP6b2。pUC57-1879-RP6b2AndpUC57-1879RP02the related structures of the pegRNA are the same, and the PBS sequences are different.

PCR amplification was carried out using primers mAmp-BsF/tMet-BsF (i.e., the primer pair consisting of mAmp-BsF and tMet-BsF in Table 1) and primer HDV-BSR2/mAmp-BSR (i.e., the primer pair consisting of HDV-BSR2 and mAmp-BSR in Table 1), respectively, with the synthetic vector pUC57-1879RP7b (pUC57-1879RP7b is a cloning vector consisting of 1879RP7b fragment shown as sequence 12 in the sequence Listing and inserted in reverse direction into lacZ of pUC57-simple, and the reverse transcription template and primer binding site sequence contained in this fragment are shown as 1879RP7b-rtT/PBS in Table 1), and with primer HDV-BsF2The primer pair consisting of/tMet-BSR (HDV-BsF 2 and tMet-BSR in the table 1) takes a synthetic segment OsWS2 (the sequence of which is shown as the sequence 2 in the sequence table) as a template for PCR amplification. Mixing and recovering the obtained three PCR products, performing enzyme digestion by BsaI, purifying and recovering the enzyme digestion product again to obtain a purified enzyme digestion product, performing self-connection on the obtained purified enzyme digestion product, and naming the obtained recombinant vector with a correct sequence as a recombinant vectorpUC57-1879-RP7b2。pUC57-1879-RP7b2AndpUC57-1879RP02the related structures of the pegRNA are the same, and the PBS sequences are different.

Wherein, the 3 rd-8 th position of the sequence 5 is a BsaI recognition sequence, the 14 th-33 th position is a Spacer sequence, the 34 th-109 th position is a sgRNA framework sequence, the 110 th position along with charge 132 is a rtT sequence, the 133 th position along with charge 145 is a PBS sequence, the 146 th position along with charge 213 is an HDV sequence, the 222 th position along with charge 293 is a tMet sequence, the 294 th position along with charge 313 is a Spacer sequence, the 314 th position along with charge 389 is a sRNA framework sequence, the 390 th position along with charge 412 is a rtT sequence, the 413 th position along with charge 425 is a PBS sequence, and the 431 th position along with charge 436 is a BsaI sequence recognition sequence.

Sequences 6-12 are the sequences obtained by sequentially replacing PBS at positions 133-145 and 413-425 of sequence 5 with "CAAGTCgATCTTC", "CAAGTCgATCTTg", "CAAGTCgATCTag", "CAAGTCgATCaag", "CAAGTCCATCaag", "CAAGTCCATCTag" and "CAAGTCCATCTTg", respectively, from "CAAGTCCATCTTC", and FIG. 2 is shown.

2. Constructing a U3 promoter-driven lead editing binary vector containing the pegRNA: establishing a Golden Gate reaction containing BsaI, and the specific process is as follows:

the vector backbone obtained was ligated to pUC57-1879RP02 obtained in step 1 of BsaI digestion by BsaI digestion of pG3H-PE2-U3(Jiang et al genome Biology (2020)21:257, primer digestion genes W542L and S621I double digestion in two ALS genes in mail), and the resulting recombinant vector with the correct sequence was named pUC57-1879RP02p1879-RP02. The structural features of the pegRNA expression cassette in p1879-RP02 are as follows: OsU3p-tGly-pegR1-HDV-OsU3t-TaU3p-tMet-pegR2-HDV-TaU3 t. The structural features of p1879-RP02 are as follows: RB-OsU3p-pegR-OsU3t-TaU3p-pegR-TaU3t-Ubi1p-Cas9n-RT-E9 t-Hyg-LB.

The BsaI is used for digesting pG3H-PE2-U3, and the obtained vector framework and the pUC57-1 obtained in the step 1 are combined879-RP1b2 were ligated by digesting with BsaI, and the resulting recombinant vector with the correct sequence was namedp1879-RP1b2。

Digesting pG3H-PE2-U3 by BsaI, and mixing the obtained vector skeleton with the vector skeleton obtained in the step 1pUC57-1879-RP2b2The fragments obtained by BsaI enzyme digestion are connected, and the obtained recombinant vector with correct sequence is named asp1879-RP2b2。

The BsaI is used for enzyme digestion of pG3H-PE2-U3, the obtained vector framework is connected with the fragment obtained by the enzyme digestion of pUC57-1879-RP3b2 obtained in the step 1 through the BsaI, and the obtained recombinant vector with the correct sequence is named as the recombinant vectorp1879-RP3b2。

The BsaI is used for enzyme digestion of pG3H-PE2-U3, the obtained vector framework is connected with the fragment obtained by the enzyme digestion of pUC57-1879-RP4b2 obtained in the step 1 through the BsaI, and the obtained recombinant vector with the correct sequence is named as the recombinant vectorp1879-RP4b2。

The BsaI is used for enzyme digestion of pG3H-PE2-U3, the obtained vector framework is connected with the fragment obtained by the enzyme digestion of pUC57-1879-RP5b2 obtained in the step 1 through the BsaI, and the obtained recombinant vector with the correct sequence is named as the recombinant vectorp1879-RP5b2。

The BsaI is used for enzyme digestion of pG3H-PE2-U3, the obtained vector framework is connected with the fragment obtained by the enzyme digestion of pUC57-1879-RP6b2 obtained in the step 1 through the BsaI, and the obtained recombinant vector with the correct sequence is named as the recombinant vectorp1879-RP6b2。

The BsaI is used for enzyme digestion of pG3H-PE2-U3, the obtained vector framework is connected with the fragment obtained by the enzyme digestion of pUC57-1879-RP7b2 obtained in the step 1 through the BsaI, and the obtained recombinant vector with the correct sequence is named as the recombinant vectorp1879-RP7b2。

Second, 35U6 fusion type promoter driving the construction of the expression vector of the pegRNA

1. Construction of a 35U6 fusion promoter-driven, lead-editing binary vector containing the pegRNA: establishing a Golden Gate reaction containing BsaI, and specifically comprising the following steps:

using the Golden Gate reaction, pG3H-PE2-35C (Jiang et al. Genom) was replaced by a DNA fragment between two BsaI cleavage sites of a pegR1-HDV-tMet-pegR2 fragment (i.e., 1879RP0 fragment as described above, whose sequence is shown as sequence 5 in the sequence listing) containing a target of I1879V, a reverse transcription template, and the likee Biology (2020)21:257, small DNA fragments between two BsaI cleavage sites of Prime editing effective genes W542L and S621I double mutations in two ALS genes in main), and the obtained recombinant vector with correct sequence is named asp1879-RP0. The structural features of the pegRNA expression cassette of p1879-RP0 are as follows: 35S-CmYLCV-U6-tGly-pegR1-HDV-tMet-pegR 2-HDV-polyT-HSPt. The p1879-RP0 structural features are as follows: RB-35U6p-pegRNA-HSPt-Ubi1p-Cas9n-RT-E9 t-Hyg-LB.

Similarly, by using Golden Gate reaction, the DNA fragment between two BsaI cleavage sites of PBS fragment 1879RP1b, 1879RP2b, 1879RP3b, 1879RP4b and 1879RP5b containing different mutant nucleotides is replaced by the small DNA fragment between two BsaI cleavage sites of pG3H-PE2-35C, and the obtained recombinant vectors with correct sequences are named as the recombinant vectors respectivelyp1879-RP1b、p1879-RP2b、p1879-RP3b、p1879-RP4bAndp1879-RP5b。

wherein, the physical map of pG3H-PE2-35C vector T-DNA containing the pegRNA is shown in the upper part of FIG. 1. LB and RB are left and right borders of T-DNA respectively, 35S-CmYLCV-U6p is a fusion promoter of 35Sen-CmYLCV and U6, 35Sen is CaMV35S enhancer, 35Sen-CmYLCV is a fusion polymerase II promoter, U6 is a truncated U6-26 polymerase III promoter, and the expression cassette of the pegRNA gene is shown in the figure and sequence 3 in the sequence table. ZmUbi1p is maize Ubi1 promoter, Cas9H840A is codon optimized SpCas9 nickase (H840A); RT is optimized reverse transcriptase (M _ MLV), E9t is pea rbcS-E9 terminator; hyg is the hygromycin resistance gene. The Cas9H840A expression box sequence is shown as sequence 1 in the sequence table.

In the sequence 3, the sequence of 35Sen at positions 1-435, the sequence of CmYLCV at positions 436-898, i.e., the fusion polymerase II promoter at positions 1-898, the truncated U6-26 polymerase III promoter sequence at positions 907-994, the tGly sequence at positions 1002-1072, the Spacer sequence at positions 1073-1092, the sgRNA framework sequence at positions 1093-1168, the rtT-PBS sequence at positions 1169-1197, the HDV sequence at positions 1198-1265, the tMet sequence at positions 1274-1345, the Spacer sequence at positions 1346-1365, the sgRNA sequence at positions 1366-1441, the rtT-PBS sequence at positions 1442-1470, the HDT-PBS sequence at positions 1538 and the HSPT-1817 sequences at positions 1539-1819.

The physical map of pG3H-PE2-U3 vector T-DNA containing the pegRNA is shown in the lower part of figure 1, OsU3p is a promoter, and the expression frame of the pegRNA gene is shown in the figure and a sequence 4 in a sequence table; ZmUbi1p is maize Ubi1 promoter, Cas9H840A is codon optimized SpCas9 nickase (H840A) gene; RT is optimized reverse transcriptase (M _ MLV), E9t is pea rbcS-E9 terminator; hyg is the hygromycin resistance gene.

In the sequence 4, the 1-437 is OsU3p sequence, the 444-514 is tGly sequence, the 515-534 is Spacer sequence, the 535-610 is sgRNA framework sequence, the 611-639 is rtT-PBS sequence, the 640-707 is HDV sequence, the 708-801 is OsU3t sequence, the 811-1334 is TaU3p sequence, the 1344-1415 is tMet sequence, the 1416-1435 is Spacer sequence, the 1436-1511 is sgRNA framework sequence, the 1512-PBS 1540 is rtT-1540 sequence, the 1541-1608 is HDV sequence, and the 1609-1667 is TaU3t sequence.

Example 2 transformation of Rice protoplasts and analysis of efficiency of Pilot editing

1. The vector is subjected to 3 independent rice protoplast transformation experiments by a PEG-mediated protoplast transformation method by using a leader-editing binary vector for driving the pegRNA by 6U 6 fusion promoters such as p1879-RP0, p1879-RP1b and the like and a leader-editing binary vector for driving the pegRNA by 6U 3 promoters such as p1879-RP02, p1879-RP1b2 and the like in a Wiger-Straus kit. Protoplasts are derived from Nipponbare.

2. Analyzing the mutation condition of the gene OsACC in the rice protoplast, carrying out PCR amplification on a fragment containing a target point by taking I1879V-F/I1879V-R as a primer and taking the transformed rice protoplast genome as a template, carrying out high-throughput sequencing on the fragment, and analyzing the mutation condition.

Protoplast editing efficiency by high throughput sequencing analysis, as shown in table 2 and fig. 3, the promoter 35U6 driven or U3 driven the expression of the pegRNA, and the mutation efficiency of the vector containing 1879RP1b fragment was higher.

Example 3 Stable transformation of Rice and Pilot editing efficiency analysis

1. Vectors driven by 8U 3 promoters such as vectors p1879-RP02, p1879-RP1b2 and p1879-RP2b2 are respectively introduced into engineered Agrobacterium LBA4404(Zhang, Zhang et al 2019, A Novel corner Vector System Unit with genetic Genes Enhances CRISPR/Cas Delivery in Maize) containing auxiliary Vector pVS1-VIR2 to form strains containing Ternary Vector systems. The agrobacterium strain is used for transforming 11 varieties of rice midflowers, and a transgenic strain is screened by utilizing 50mg/L hygromycin.

2. Analyzing the mutation condition of the gene OsACC in the rice transgenic line, carrying out PCR amplification on a segment containing a target point by taking I1879V-IDF/I1879V-IDR as a primer and taking the rice transgenic line genome as a template, carrying out Sanger sequencing by taking I1879V-IDF as a detection primer, and detecting the mutation condition.

The stable transformation results of rice are shown in Table 3, 1 of 37 transgenic lines of the transformation control vector p1879-RP02 is accurately edited, and the editing efficiency is 2.7% (1/37); the efficiency of the editing of 4 experimental groups was improved compared to the control vector (p1879-RP 02). Wherein, in 36 transgenic strains of p1879-RP1b2, 3 strains are accurately edited, the accurate editing efficiency is 8.3% (3/36), which is about 3 times of that of the control group; in 36 transgenic strains of p1879-RP5b2, accurate editing occurs in 4 strains, the accurate editing efficiency is 11.1% (4/36), which is about 4 times of the efficiency of a control group, and simultaneously, non-accurate editing occurs in 1 strain, namely, the total editing efficiency of the vector p1879-RP5b2 is 13.9% (5/36); the total editing efficiency of p1879-RP6b2 was 9.5%, and the total gene editing efficiency of p1879-RP7b2 was 6.9%, both significantly higher than the control.

The experimental result shows that the editing efficiency of the pilot editing system in the plant can be effectively improved by introducing the mutant base in the middle position of the PBS or introducing the mutant base at the 3' end of the PBS.

TABLE 2 high throughput method for analyzing efficiency of accurate editing in protoplasts

Note: the average is the average of the data from 3 independent transformation experiments (n ═ 1,2,3)

TABLE 3 Pilot editing efficiency of Rice transgenic lines

Note: the table is statistically calculated according to the direct sequencing results of the PCR products of the transgenic lines.

As used herein, the term "exact editing" refers to the mutation of the codon 1879 of OsACC from ATA to GTA and the mutation of G-c, G-c introduced for the introduction of a cleavage site.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> university of agriculture in China

<120> a carrier and method for improving efficiency of pilot editing

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 8998

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

agtgcagcgt gacccggtcg tgcccctcac tagagataat gagcattgca tgtctaagtt 60

ataaaaaatt accacatatt ttttttgtca cacttgtttg aagtgcagtt tatctatctt 120

tatacatata tttaaacttt actctacgaa taatataatc tatagtacta caataatatc 180

agtgttttag agaatcatat aaatgaacag ttagacatgg tctaaaggac aattgagtat 240

tttgacaaca ggactctaca gttttatctt tttagtgtgc atgtgttctc cttttttttt 300

gcaaatagct tcacctatat aatacttcat ccattttatt agtacatcca tttagggttt 360

agggttaatg gtttttatag actaattttt ttagtacatc tattttattc tattttagcc 420

tctaaattaa gaaaactaaa actctatttt agttttttta tttaataatt tagatataaa 480

atagaataaa ataaagtgac taaaaattaa acaaataccc tttaagaaat taaaaaaact 540

aaggaaacat ttttcttgtt tcgagtagat aatgccagcc tgttaaacgc cgtcgacgag 600

tctaacggac accaaccagc gaaccagcag cgtcgcgtcg ggccaagcga agcagacggc 660

acggcatctc tgtcgctgcc tctggacccc tctcgagagt tccgctccac cgttggactt 720

gctccgctgt cggcatccag aaattgcgtg gcggagcggc agacgtgagc cggcacggca 780

ggcggcctcc tcctcctctc acggcaccgg cagctacggg ggattccttt cccaccgctc 840

cttcgctttc ccttcctcgc ccgccgtaat aaatagacac cccctccaca ccctctttcc 900

ccaacctcgt gttgttcgga gcgcacacac acacaaccag atctccccca aatccacccg 960

tcggcacctc cgcttcaagg tacgccgctc gtcctccccc cccccccctc tctaccttct 1020

ctagatcggc gttccggtcc atgcttaggg cccggtagtt ctacttctgt tcatgtttgt 1080

gttagatccg tgtttgtgtt agatccgtgc tgctagcgtt cgtacacgga tgcgacctgt 1140

acgtcagaca cgttctgatt gctaacttgc cagtgtttct ctttggggaa tcctgggatg 1200

gctctagccg ttccgcagac gggatcgatt tcatgatttt ttttgtttcg ttgcataggg 1260

tttggtttgc ccttttcctt tatttcaata tatgccgtgc acttgtttgt cgggtcatct 1320

tttcatgctt ttttttgtct tggttgtgat gatgtggtct ggttgggcgg tcgttctaga 1380

tcggagtaca attctgtttc aaactacctg gtggatttat taattttgga tctgtatgtg 1440

tgtgccatac atattcatag ttacgaattg aagatgatgg atggaaatat cgatctagga 1500

taggtataca tgttgatgcg ggttttactg atgcatatac agagatgctt tttgttcgct 1560

tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc gttctagatc ggagtagaat 1620

actgtttcaa actacctggt gtatttatta attttggaac tgtatgtgtg tgtcatacat 1680

cttcatagtt acgagtttaa gatggatgga aatatcgatc taggataggt atacatgttg 1740

atgtgggttt tactgatgca tatacatgat ggcatatgca gcatctattc atatgctcta 1800

accttgagta cctatctatt ataataaaca agtatgtttt ataattattt tgatcttgat 1860

atacttggat gatggcatat gcagcagcta tatgtggatt tttttagccc tgccttcata 1920

cgctatttat ttgcttggta ctgtttcttt tgtcgatgct caccctgttg tttggtgtta 1980

cttctgcagg tacctaggcc tctagatgaa gaggacagcc gatggcagcg agttcgagag 2040

ccctaagaag aagaggaagg tggacaagaa gtactcgatc ggcctcgata ttgggactaa 2100

ctctgttggc tgggccgtga tcaccgacga gtacaaggtg ccctcaaaga agttcaaggt 2160

cctgggcaac accgatcggc attccatcaa gaagaatctc attggcgctc tcctgttcga 2220

cagcggcgag acggctgagg ctacgcggct caagcgcacc gcccgcaggc ggtacacgcg 2280

caggaagaat cgcatctgct acctgcagga gattttctcc aacgagatgg cgaaggttga 2340

cgattctttc ttccacaggc tggaggagtc attcctcgtg gaggaggata agaagcacga 2400

gcggcatcca atcttcggca acattgtcga cgaggttgcc taccacgaga agtaccctac 2460

gatctaccat ctgcggaaga agctcgtgga ctccacagat aaggcggacc tccgcctgat 2520

ctacctcgct ctggcccaca tgattaagtt caggggccat ttcctgatcg agggggatct 2580

caacccggac aatagcgatg ttgacaagct gttcatccag ctcgtgcaga cgtacaacca 2640

gctcttcgag gagaacccca ttaatgcgtc aggcgtcgac gcgaaggcta tcctgtccgc 2700

taggctctcg aagtctcggc gcctcgagaa cctgatcgcc cagctgccgg gcgagaagaa 2760

gaacggcctg ttcgggaatc tcattgcgct cagcctgggg ctcacgccca acttcaagtc 2820

gaatttcgat ctcgctgagg acgccaagct gcagctctcc aaggacacat acgacgatga 2880

cctggataac ctcctggccc agatcggcga tcagtacgcg gacctgttcc tcgctgccaa 2940

gaatctgtcg gacgccatcc tcctgtctga tattctcagg gtgaacaccg agattacgaa 3000

ggctccgctc tcagcctcca tgatcaagcg ctacgacgag caccatcagg atctgaccct 3060

cctgaaggcg ctggtcaggc agcagctccc cgagaagtac aaggagatct tcttcgatca 3120

gtcgaagaac ggctacgctg ggtacattga cggcggggcc tctcaggagg agttctacaa 3180

gttcatcaag ccgattctgg agaagatgga cggcacggag gagctgctgg tgaagctcaa 3240

tcgcgaggac ctcctgagga agcagcggac attcgataac ggcagcatcc cacaccagat 3300

tcatctcggg gagctgcacg ctatcctgag gaggcaggag gacttctacc ctttcctcaa 3360

ggataaccgc gagaagatcg agaagattct gactttcagg atcccgtact acgtcggccc 3420

actcgctagg ggcaactccc gcttcgcttg gatgacccgc aagtcagagg agacgatcac 3480

gccgtggaac ttcgaggagg tggtcgacaa gggcgctagc gctcagtcgt tcatcgagag 3540

gatgacgaat ttcgacaaga acctgccaaa tgagaaggtg ctccctaagc actcgctcct 3600

gtacgagtac ttcacagtct acaacgagct gactaaggtg aagtatgtga ccgagggcat 3660

gaggaagccg gctttcctgt ctggggagca gaagaaggcc atcgtggacc tcctgttcaa 3720

gaccaaccgg aaggtcacgg ttaagcagct caaggaggac tacttcaaga agattgagtg 3780

cttcgattcg gtcgagatct ctggcgttga ggaccgcttc aacgcctccc tggggaccta 3840

ccacgatctc ctgaagatca ttaaggataa ggacttcctg gacaacgagg agaatgagga 3900

tatcctcgag gacattgtgc tgacactcac tctgttcgag gaccgggaga tgatcgagga 3960

gcgcctgaag acttacgccc atctcttcga tgacaaggtc atgaagcagc tcaagaggag 4020

gaggtacacc ggctggggga ggctgagcag gaagctcatc aacggcattc gggacaagca 4080

gtccgggaag acgatcctcg acttcctgaa gagcgatggc ttcgcgaacc gcaatttcat 4140

gcagctgatt cacgatgaca gcctcacatt caaggaggat atccagaagg ctcaggtgag 4200

cggccagggg gactcgctgc acgagcatat cgcgaacctc gctggctcgc cagctatcaa 4260

gaaggggatt ctgcagaccg tgaaggttgt ggacgagctg gtgaaggtca tgggcaggca 4320

caagcctgag aacatcgtca ttgagatggc ccgggagaat cagaccacgc agaagggcca 4380

gaagaactca cgcgagagga tgaagaggat cgaggagggc attaaggagc tggggtccca 4440

gatcctcaag gagcacccgg tggagaacac gcagctgcag aatgagaagc tctacctgta 4500

ctacctccag aatggccgcg atatgtatgt ggaccaggag ctggatatta acaggctcag 4560

cgattacgac gtcgatgcca tcgttccaca gtcattcctg aaggatgact ccattgacaa 4620

caaggtcctc accaggtcgg acaagaaccg gggcaagtct gataatgttc cttcagagga 4680

ggtcgttaag aagatgaaga actactggcg ccagctcctg aatgccaagc tgatcacgca 4740

gcggaagttc gataacctca caaaggctga gaggggcggg ctctctgagc tggacaaggc 4800

gggcttcatc aagaggcagc tggtcgagac acggcagatc actaagcacg ttgcgcagat 4860

tctcgactca cggatgaaca ctaagtacga tgagaatgac aagctgatcc gcgaggtgaa 4920

ggtcatcacc ctgaagtcaa agctcgtctc cgacttcagg aaggatttcc agttctacaa 4980

ggttcgggag atcaacaatt accaccatgc ccatgacgcg tacctgaacg cggtggtcgg 5040

cacagctctg atcaagaagt acccaaagct cgagagcgag ttcgtgtacg gggactacaa 5100

ggtttacgat gtgaggaaga tgatcgccaa gtcggagcag gagattggca aggctaccgc 5160

caagtacttc ttctactcta acattatgaa tttcttcaag acagagatca ctctggccaa 5220

tggcgagatc cggaagcgcc ccctcatcga gacgaacggc gagacggggg agatcgtgtg 5280

ggacaagggc agggatttcg cgaccgtcag gaaggttctc tccatgccac aagtgaatat 5340

cgtcaagaag acagaggtcc agactggcgg gttctctaag gagtcaattc tgcctaagcg 5400

gaacagcgac aagctcatcg cccgcaagaa ggactgggat ccgaagaagt acggcgggtt 5460

cgacagcccc actgtggcct actcggtcct ggttgtggcg aaggttgaga agggcaagtc 5520

caagaagctc aagagcgtga aggagctgct ggggatcacg attatggagc gctccagctt 5580

cgagaagaac ccgatcgatt tcctggaggc gaagggctac aaggaggtga agaaggacct 5640

gatcattaag ctccccaagt actcactctt cgagctggag aacggcagga agcggatgct 5700

ggcttccgct ggcgagctgc agaaggggaa cgagctggct ctgccgtcca agtatgtgaa 5760

cttcctctac ctggcctccc actacgagaa gctcaagggc agccccgagg acaacgagca 5820

gaagcagctg ttcgtcgagc agcacaagca ttacctcgac gagatcattg agcagatttc 5880

cgagttctcc aagcgcgtga tcctggccga cgcgaatctg gataaggtcc tctccgcgta 5940

caacaagcac cgcgacaagc caatcaggga gcaggctgag aatatcattc atctcttcac 6000

cctgacgaac ctcggcgccc ctgctgcttt caagtacttc gacacaacta tcgatcgcaa 6060

gaggtacaca agcactaagg aggtcctgga cgcgaccctc atccaccagt cgattaccgg 6120

cctctacgag acgcgcatcg acctgtctca gctcgggggc gactcaggcg gctcatcggg 6180

cgggtcaagc gggtcggaga caccgggcac atcagagagc gctacccctg agtcatcagg 6240

cggctcttca ggcggcagct caaccctgaa cattgaggac gagtaccggc tgcacgagac 6300

gagcaaggag ccagacgttt cgctcggcag cacttggctc tctgacttcc cacaggcttg 6360

ggccgagact ggcggcatgg gcctggccgt gcgccaggct ccactgatca tccctctgaa 6420

ggcgacctcc accccggttt ctattaagca gtacccgatg agccaggagg ccaggctggg 6480

gatcaagcca cacattcagc ggctgctgga ccagggcatc ctggtgccat gccagtcccc 6540

gtggaatact ccgctcctgc cggtgaagaa gcctgggaca aacgactaca ggccggttca 6600

ggatctcagg gaggtgaaca agcgcgtgga ggacatccat ccgacagtgc cgaacccgta 6660

caatctgctg tcgggcctgc ctccgagcca ccagtggtac accgtcctgg acctcaagga 6720

cgctttcttc tgcctgcggc tgcacccgac gtctcagccg ctgttcgcgt tcgagtggcg 6780

cgacccagag atgggcattt ccggccagct gacctggaca cgcctacccc agggcttcaa 6840

gaactccccg actctcttca acgaggctct ccaccgggat ctcgcggact tcaggattca 6900

gcatcccgat ctgatcctgc tccagtatgt tgacgacctc ctcctggccg cgacgtcgga 6960

gctggactgc cagcagggca cccgggcgct gctgcagaca ctgggcaatc tggggtaccg 7020

cgcctctgcg aagaaggcgc agatctgcca gaagcaagtg aagtacctgg gctacctcct 7080

gaaggagggc cagcgctggc tcactgaggc gaggaaggag actgttatgg gccagcccac 7140

tccaaagact ccgaggcagc tcagggagtt cctcggcaag gctgggttct gccgcctgtt 7200

catccctggg ttcgctgaga tggctgcgcc gctctacccg ctgactaagc cggggacact 7260

gttcaactgg gggccagacc agcagaaggc gtaccaggag attaagcagg cgctgctgac 7320

ggccccagcg ctcggcctac cagacctgac gaagccgttc gagctgttcg ttgacgagaa 7380

gcaggggtac gcgaagggcg tgctgacaca gaagctgggg ccttggcgcc gcccggtcgc 7440

gtacctgtcg aagaagctgg acccagtcgc tgctgggtgg cctccatgcc tccggatggt 7500

cgctgctatt gcggttctga ccaaggatgc ggggaagctc acaatggggc agcctctcgt 7560

gatcctggct ccacatgcgg tggaggcgct ggtgaagcag ccaccggacc ggtggctgtc 7620

gaacgctcgg atgacacact accaggcgct cctcctcgat acagaccggg ttcagttcgg 7680

gcctgtggtt gctctgaacc cagccacact gctgccactc cctgaggagg gcctccagca 7740

caattgcctc gacatcctgg ctgaggcgca cggcacccgc cctgatctca ccgaccagcc 7800

tctgccagat gctgaccaca cctggtacac ggatgggtcc tcgctgctgc aggagggcca 7860

gaggaaggcg ggcgccgccg tcaccacaga gacagaggtt atttgggcca aggccctacc 7920

ggctggcacc agcgcccagc gcgctgagct gatcgcgctg actcaggcgc tgaagatggc 7980

cgaggggaag aagctcaatg tttacaccga ctcgcggtac gcgttcgcta cagctcacat 8040

tcatggggag atctaccgcc ggcgcgggtg gctgacttcg gagggcaagg agattaagaa 8100

taaggacgag atcctggccc tgctcaaggc gctgttcctg ccgaagcgcc tctcaatcat 8160

tcactgcccg ggccaccaga agggccattc ggccgaggct aggggcaatc ggatggctga 8220

ccaggcggcg cggaaggcgg ctatcaccga gactcccgat acatctaccc tcctgatcga 8280

gaactcgagc ccaagcggcg ggagcaagcg gactgcggat gggtctgagt tcgagccaaa 8340

gaagaagagg aaggtgtgag ctcagagctt tcgttcgtat catcggtttc gacaacgttc 8400

gtcaagttca atgcatcagt ttcattgcgc acacaccaga atcctactga gtttgagtat 8460

tatggcattg ggaaaactgt ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt 8520

ttttattcgg ttttcgctat cgaactgtga aatggaaatg gatggagaag agttaatgaa 8580

tgatatggtc cttttgttca ttctcaaatt aatattattt gttttttctc ttatttgttg 8640

tgtgttgaat ttgaaattat aagagatatg caaacatttt gttttgagta aaaatgtgtc 8700

aaatcgtggc ctctaatgac cgaagttaat atgaggagta aaacacttgt agttgtacca 8760

ttatgcttat tcactaggca acaaatatat tttcagacct agaaaagctg caaatgttac 8820

tgaatacaag tatgtcctct tgtgttttag acatttatga actttccttt atgtaatttt 8880

ccagaatcct tgtcagattc taatcattgc tttataatta tagttatact catggatttg 8940

tagttgagta tgaaaatatt ttttaatgca ttttatgact tgccaattga ttgacaac 8998

<210> 2

<211> 1050

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

ttggtctcat gcagggtatg gtggtgcaat ggggttttag agctagaaat agcaagttaa 60

aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgca aacctatctt 120

ctaattgcac caccatggcc ggcatggtcc cagcctcctc gctggcgccg gctgggcaac 180

atgcttcggc atggcgaatg ggactttttt ttttcgtttt gcattgagtt ttctccgtcg 240

catgtttgca gttttatttt ccgttttgca ttgaaatttc tccgtctcat gtttgcagcg 300

tgttcaacat gaatccaaac cacacggagt tcaaattccc acagattaag gctcgtccgt 360

cgcacaaggt aatgtgtgaa tattatatct gtcgtgcaaa attgcctggc ctgcacaatt 420

gctgttatag ttggcggcag ggagagtttt aacattgact agcgtgctga taatttgtga 480

gaaataataa ttgacaagta gatactgaca tttgagaaga gcttctgaac tgttattagt 540

aacaaaaatg gaaagctgat gcacggaaaa aggaaagaaa aagccatact tttttttagg 600

taggaaaaga aaaagccata cgagactgat gtctctcaga tgggccggga tctgtctatc 660

tagcaggcag cagcccacca acctcacggg ccagcaatta cgagtccttc taaaagctcc 720

cgccgagggg cgctggcgct gctgtgcagc agcacgtcta acattagtcc cacctcgcca 780

gtttacaggg agcagaacca gcttataagc ggaggcgcgg caccaagaag cgaacaacaa 840

atcagagtgg cgcagcggaa gcgtggtggg cccataaccc acaggtccca ggatcgaaac 900

ctggctctga taccttgaat gcgcccccac ttgttttaga gctagaaata gcaagttaaa 960

ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctg cctatgatac 1020

caattggggg cgcattcggc cagagaccaa 1050

<210> 3

<211> 1817

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> misc_feature

<222> (1073)..(1092)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1169)..(1197)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1346)..(1365)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1442)..(1470)

<223> n is a, c, g, or t

<400> 3

atggagtcaa agattcaaat agaggaccta acagaactcg ccgtaaagac tggcgaacag 60

ttcatacaga gtctcttacg actcaatgac aagaagaaaa tcttcgtcaa catggtggag 120

cacgacacac ttgtctactc caaaaatatc aaagatacag tctcagaaga ccaaagggca 180

attgagactt ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca ttgcccagct 240

atctgtcact ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa atgccatcat 300

tgcgataaag gaaaggccat cgttgaagat gcctctgccg acagtggtcc caaagatgga 360

cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa 420

gtggattgat gtgattggca gacatactgt cccacaaatg aagatggaat ctgtaaaaga 480

aaacgcgtga aataatgcgt ctgacaaagg ttaggtcggc tgcctttaat caataccaaa 540

gtggtcccta ccacgatgga aaaactgtgc agtcggtttg gctttttctg acgaacaaat 600

aagattcgtg gccgacaggt gggggtccac catgtgaagg catcttcaga ctccaataat 660

ggagcaatga cgtaagggct tacgaaataa gtaagggtag tttgggaaat gtccactcac 720

ccgtcagtct ataaatactt agcccctccc tcattgttaa gggagcaaaa tctcagagag 780

atagtcctag agagagaaag agagcaagta gcctagaagt agtcaaggcg gcgaagtatt 840

caggcacgtg gccaggaaga agaaaagcca agacgacgaa aacaggtaag agctaagcat 900

ctagataagt tgaaaacaat cttcaaaagt cccacatcgc ttagataaga aaacgaagct 960

gagtttatat acagctagag tcgaagtagt gattgaacaa agcaccagtg gtctagtggt 1020

agaatagtac cctgccacgg tacagacccg ggttcgattc ccggctggtg cannnnnnnn 1080

nnnnnnnnnn nngttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca 1140

acttgaaaaa gtggcaccga gtcggtgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnggc 1200

cggcatggtc ccagcctcct cgctggcgcc ggctgggcaa catgcttcgg catggcgaat 1260

gggacaacaa caaatcagag tggcgcagcg gaagcgtggt gggcccataa cccacaggtc 1320

ccaggatcga aacctggctc tgatannnnn nnnnnnnnnn nnnnngtttt agagctagaa 1380

atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 1440

cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ggccggcatg gtcccagcct cctcgctggc 1500

gccggctggg caacatgctt cggcatggcg aatgggactt ttttttgata tctccggggc 1560

taattgaata tgaagatgaa gatgaaatat ttggtgtgtc aaataaaaag ctggtgtgct 1620

taagtttgtg tttttttctt ggcttgttgt gttatgaatt tgtggctttt tctaatatta 1680

aatgaatgta agatctcatt ataatgaata aacaaatgtt tctataatcc attgtgaatg 1740

ttttgttgga tctcttctgc agcatataac tactgtatgt gctatggtat ggactatgga 1800

atatgattaa agataag 1817

<210> 4

<211> 1667

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> misc_feature

<222> (515)..(534)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (611)..(639)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1416)..(1435)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1512)..(1540)

<223> n is a, c, g, or t

<400> 4

agtaattcat ccaggtcacc aagttctagg attttcagaa ctgcaactta ttttatcaag 60

gaatctttaa acatacgaac agatcactta aagttcttct gaagcaactt aaagttatca 120

ggcttgcatg gatcttggag gaatcagatg tgcagtcagg gaccatagca caagacaggc 180

gtcttctact ggtgctacca gcaaatgctg gaagccggga acactgggta cgttggaaac 240

cacgtgatgt gaagaagtaa gataaactgt aggagaaaag catttcgtag tgggccatga 300

agcctttcag gacatgtatt gcagtatggg ccggcccatt acgcaattgg acgacaacaa 360

agtctagtat tagtaccacc tcggctatcc acatagatca aagctgattt aaaagagttg 420

tgcagatgat ccgtggcaac aaagcaccag tggtctagtg gtagaatagt accctgccac 480

ggtacagacc cgggttcgat tcccggctgg tgcannnnnn nnnnnnnnnn nnnngtttta 540

gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc 600

gagtcggtgc nnnnnnnnnn nnnnnnnnnn nnnnnnnnng gccggcatgg tcccagcctc 660

ctcgctggcg ccggctgggc aacatgcttc ggcatggcga atgggacttt tttttttcgt 720

tttgcattga gttttctccg tcgcatgttt gcagttttat tttccgtttt gcattgaaat 780

ttctccgtct catgtttgca gcgtgttcaa catgaatcca aaccacacgg agttcaaatt 840

cccacagatt aaggctcgtc cgtcgcacaa ggtaatgtgt gaatattata tctgtcgtgc 900

aaaattgcct ggcctgcaca attgctgtta tagttggcgg cagggagagt tttaacattg 960

actagcgtgc tgataatttg tgagaaataa taattgacaa gtagatactg acatttgaga 1020

agagcttctg aactgttatt agtaacaaaa atggaaagct gatgcacgga aaaaggaaag 1080

aaaaagccat actttttttt aggtaggaaa agaaaaagcc atacgagact gatgtctctc 1140

agatgggccg ggatctgtct atctagcagg cagcagccca ccaacctcac gggccagcaa 1200

ttacgagtcc ttctaaaagc tcccgccgag gggcgctggc gctgctgtgc agcagcacgt 1260

ctaacattag tcccacctcg ccagtttaca gggagcagaa ccagcttata agcggaggcg 1320

cggcaccaag aagcgaacaa caaatcagag tggcgcagcg gaagcgtggt gggcccataa 1380

cccacaggtc ccaggatcga aacctggctc tgatannnnn nnnnnnnnnn nnnnngtttt 1440

agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac 1500

cgagtcggtg cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ggccggcatg gtcccagcct 1560

cctcgctggc gccggctggg caacatgctt cggcatggcg aatgggactt ttttttttgt 1620

ccttctgttt ttttagtcag tctctttttt cagaagtaca acatctt 1667

<210> 5

<211> 438

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

ttggtctcat gcacaaggaa gatggacttg gtggttttag agctagaaat agcaagttaa 60

aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgca cttccatgta 120

cattgtcgac accaagtcca tcttcggccg gcatggtccc agcctcctcg ctggcgccgg 180

ctgggcaaca tgcttcggca tggcgaatgg gacaacaaca aatcagagtg gcgcagcgga 240

agcgtggtgg gcccataacc cacaggtccc aggatcgaaa cctggctctg atacaaggaa 300

gatggacttg gtggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc 360

aacttgaaaa agtggcaccg agtcggtgca cttccatgta cattgtcgac accaagtcca 420

tcttcggcca gagaccaa 438

<210> 6

<211> 438

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

ttggtctcat gcacaaggaa gatggacttg gtggttttag agctagaaat agcaagttaa 60

aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgca cttccatgta 120

cattgtcgac accaagtcga tcttcggccg gcatggtccc agcctcctcg ctggcgccgg 180

ctgggcaaca tgcttcggca tggcgaatgg gacaacaaca aatcagagtg gcgcagcgga 240

agcgtggtgg gcccataacc cacaggtccc aggatcgaaa cctggctctg atacaaggaa 300

gatggacttg gtggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc 360

aacttgaaaa agtggcaccg agtcggtgca cttccatgta cattgtcgac accaagtcga 420

tcttcggcca gagaccaa 438

<210> 7

<211> 438

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

ttggtctcat gcacaaggaa gatggacttg gtggttttag agctagaaat agcaagttaa 60

aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgca cttccatgta 120

cattgtcgac accaagtcga tcttgggccg gcatggtccc agcctcctcg ctggcgccgg 180

ctgggcaaca tgcttcggca tggcgaatgg gacaacaaca aatcagagtg gcgcagcgga 240

agcgtggtgg gcccataacc cacaggtccc aggatcgaaa cctggctctg atacaaggaa 300

gatggacttg gtggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc 360

aacttgaaaa agtggcaccg agtcggtgca cttccatgta cattgtcgac accaagtcga 420

tcttgggcca gagaccaa 438

<210> 8

<211> 438

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

ttggtctcat gcacaaggaa gatggacttg gtggttttag agctagaaat agcaagttaa 60

aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgca cttccatgta 120

cattgtcgac accaagtcga tctagggccg gcatggtccc agcctcctcg ctggcgccgg 180

ctgggcaaca tgcttcggca tggcgaatgg gacaacaaca aatcagagtg gcgcagcgga 240

agcgtggtgg gcccataacc cacaggtccc aggatcgaaa cctggctctg atacaaggaa 300

gatggacttg gtggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc 360

aacttgaaaa agtggcaccg agtcggtgca cttccatgta cattgtcgac accaagtcga 420

tctagggcca gagaccaa 438

<210> 9

<211> 438

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

ttggtctcat gcacaaggaa gatggacttg gtggttttag agctagaaat agcaagttaa 60

aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgca cttccatgta 120

cattgtcgac accaagtcga tcaagggccg gcatggtccc agcctcctcg ctggcgccgg 180

ctgggcaaca tgcttcggca tggcgaatgg gacaacaaca aatcagagtg gcgcagcgga 240

agcgtggtgg gcccataacc cacaggtccc aggatcgaaa cctggctctg atacaaggaa 300

gatggacttg gtggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc 360

aacttgaaaa agtggcaccg agtcggtgca cttccatgta cattgtcgac accaagtcga 420

tcaagggcca gagaccaa 438

<210> 10

<211> 438

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

ttggtctcat gcacaaggaa gatggacttg gtggttttag agctagaaat agcaagttaa 60

aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgca cttccatgta 120

cattgtcgac accaagtcca tcaagggccg gcatggtccc agcctcctcg ctggcgccgg 180

ctgggcaaca tgcttcggca tggcgaatgg gacaacaaca aatcagagtg gcgcagcgga 240

agcgtggtgg gcccataacc cacaggtccc aggatcgaaa cctggctctg atacaaggaa 300

gatggacttg gtggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc 360

aacttgaaaa agtggcaccg agtcggtgca cttccatgta cattgtcgac accaagtcca 420

tcaagggcca gagaccaa 438

<210> 11

<211> 438

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

ttggtctcat gcacaaggaa gatggacttg gtggttttag agctagaaat agcaagttaa 60

aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgca cttccatgta 120

cattgtcgac accaagtcca tctagggccg gcatggtccc agcctcctcg ctggcgccgg 180

ctgggcaaca tgcttcggca tggcgaatgg gacaacaaca aatcagagtg gcgcagcgga 240

agcgtggtgg gcccataacc cacaggtccc aggatcgaaa cctggctctg atacaaggaa 300

gatggacttg gtggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc 360

aacttgaaaa agtggcaccg agtcggtgca cttccatgta cattgtcgac accaagtcca 420

tctagggcca gagaccaa 438

<210> 12

<211> 438

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

ttggtctcat gcacaaggaa gatggacttg gtggttttag agctagaaat agcaagttaa 60

aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgca cttccatgta 120

cattgtcgac accaagtcca tcttgggccg gcatggtccc agcctcctcg ctggcgccgg 180

ctgggcaaca tgcttcggca tggcgaatgg gacaacaaca aatcagagtg gcgcagcgga 240

agcgtggtgg gcccataacc cacaggtccc aggatcgaaa cctggctctg atacaaggaa 300

gatggacttg gtggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc 360

aacttgaaaa agtggcaccg agtcggtgca cttccatgta cattgtcgac accaagtcca 420

tcttgggcca gagaccaa 438

Claims (10)

1. A DNA lead editing system comprises a pegRNA, wherein the pegRNA is an RNA molecule obtained by connecting a sgRNA targeting a target DNA fragment, a reverse transcription template and a primer binding site, and the 3' end of the primer binding site and/or the middle of the primer binding site are/is not matched with nucleotides of a spacer of the sgRNA.

2. The system of claim 1, wherein: one, two, or three of the three nucleotides at the 3' end of the primer binding site do not match the corresponding nucleotides of the spacer of the sgRNA; and/or the seventh nucleotide from the 3' end of the primer binding site or other sequences thereof do not match with the corresponding nucleotide of the spacer of the sgRNA.

3. The system according to claim 1 or 2, characterized in that: the DNA lead editing system also comprises a fusion protein formed by fusing Cas9 nickase (H840A) and reverse transcriptase M-MLV.

4. A vector comprising a DNA molecule that transcribes the pegRNA of claim 1 or 2.

5. The carrier of claim 4, wherein: the DNA molecule is the 515 th-639 th site of the sequence 4.

6. The carrier of claim 4 or 5, wherein: in said vector, transcription of a DNA molecule comprising a recombinant pegRNA according to claim 1 or 2 is driven by a DNA molecule represented by position 1-994 of sequence 3 of the sequence Listing or by a DNA molecule represented by position 1-437 of sequence 4 of the sequence Listing and/or by a DNA molecule represented by position 811-1334 of sequence 4 of the sequence Listing.

7. The vector according to any one of claims 4-6, wherein: the vector further comprises a gene encoding the fusion protein of claim 3.

8. Use of the system of any one of claims 1-3 or the vector of any one of claims 4-7 in gene editing.

9. A method for gene editing, comprising introducing the vector of any one of claims 4-7 into an object to be edited to achieve gene editing.

10. Use of the system of any one of claims 1 to 3 or the vector of any one of claims 4 to 7 or the method of claim 9 in plant breeding.

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