CN116064512A - Improved guide editing system and application thereof - Google Patents

Abstract

The invention relates to the field of genome editing, in particular to an improved guided editing system and application thereof. The guide editing system comprises pegRNA and a fusion protein, wherein the fusion protein consists of nCas9 (H840A) and M-MLV, and the N end of the nCas9 (H840A) is connected with T5 exonuclease or Pol-N exonuclease; the pegRNA consists of a sgRNA sequence and a PBR+RT sequence in sequence. The editing efficiency of the guiding editing system is further improved on the basis of the prior art, and the development and application of the guiding editing system are greatly promoted.

Description

Improved guide editing system and application thereof

Technical Field

The invention relates to the field of genome editing, in particular to an improved guided editing system and application thereof.

Background

The clustered regularly interspaced short palindromic repeats and their related systems (Clustered regularly interspaced short palindromic repeats associated protein, CRISPR/Cas) are widely used in animal and plant research because of their simplicity, rapidness, high efficiency, and the like, replacing almost all genome editing techniques. The CRISPR/Cas9 system mainly comprises Cas9 endonuclease protein and single-stranded guide RNA (sgRNA), wherein the Cas9 protein recognizes a specific target point under the guidance of the sgRNA, and cuts to generate DNA double-strand break (DSB) after a motif (Protospacer adjacent motif, PAM) is adjacent to a forebay, so as to trigger a self-repairing mechanism of an organism and realize DNA site-directed mutagenesis. The DSBs repair pathway common in cells is Non-homologous end joining (NHEJ) and homologous recombination (Homologous recombination, HR). HR is a precise repair pathway requiring the participation of donor templates, enabling precise site-directed mutagenesis of genes. However, HR is usually complex in vector design, mainly occurs in G2 and S phases of cell division, is limited by editing period, and has low editing efficiency, so that it is difficult to apply widely. NHEJ is an error-prone repair pathway, usually introducing only random insertions/deletions (insertion and deletion, indels) of a small range of bases, and the mutation types are diverse and difficult to control. Genome editing technologies derived from CRISPR/Cas systems include Base Editors (BE) and guide editors (PE).

Conventional BE includes a Cytosine Base Editor (CBE) and an Adenine Base Editor (ABE), mainly composed of cytosine deaminase/adenine deaminase, nCas9 (nicase Cas 9) and sgRNA. Under the guidance of sgRNA, nCas9 and deaminase fusion protein bind to the target spot to deaminize a specific base, and then base site-specific substitution is generated through DNA damage repair. Cytosine deaminase recognizes deaminated cytosine (C) to form uracil (U), which is then converted to T by DNA replication or repair, ultimately effecting the substitution of C-G to a-T base pairs. The most widely used cytosine deaminase is the rat-derived cytidine deaminase (rAPOBEC 1). Adenine deaminase recognizes adenine (A), deaminates to form inosine (I), is erroneously recognized as G in the DNA repair process because of the chemical structure of I being similar to G, and repairs T in the complementary strand as C, thereby realizing the conversion from A-T to G-C. Commonly used adenine deaminase is a heterodimer composed of optimized escherichia coli tRNA adenine deaminase (ecTadA) and wild-type ecTadA. BE can realize accurate single base mutation of target sites, but at present, two base editors can only realize C-T and A-G conversion, can not realize transversion, is easily limited by an editing window, and has limited editing types and range. Whole genome sequencing has found that BE also causes some degree of off-target mutation.

The invention and the application of PE are breakthroughs of one innovative property of genome editing technology. PE1 is composed of two parts, namely a fusion protein formed by nCas9 (H840A) and reverse transcriptase M-MLV, a primer binding sequence (Primer binding site, PBS), a reverse transcription template sequence (RT template) and a pegRNA formed by connection of the sgRNA. Under the guidance of pegRNA, the fusion protein consisting of nCas9 (H840A) and M-MLV cuts single-stranded DNA, the PBS at the 3 'end of the pegRNA is combined with the sequence at the cutting breakpoint through base complementary pairing, and reverse transcription is carried out along an RT template under the action of M-MLV, so that the 3' cohesive end containing the mutation of the target gene is synthesized. At this time, the cleavage site will simultaneously form a 3 'end containing the target edit and a 5' end containing the unedited sequence, and the 5 'end is cut and connected with the 3' end under the action of intracellular 5'-3' exonuclease, thereby realizing accurate gene site-directed mutagenesis. Based on PE1, the engineering of PE2 optimizes M-MLV protein, and increases editing efficiency relative to PE 1. Based on PE2 pegRNA, the PE3 is additionally added with the sgRNA, and the complementary DNA strand of the cutting strand is guided by the targeting pegRNA to regulate and control the intracellular DNA repair mechanism. However, the research shows that PE3 has no editing effect on a plurality of targets. PE can achieve almost any type of small fragment gene mutation, including 12 different types of base substitutions (transitions and transversions), deletions and insertions of small-range fragments, etc., and does not require donor templates and double-stranded DNA breaks.

Although the PE has strong functions, the application range is greatly limited due to low editing efficiency. Some substantial efforts, including designing pegRNA with the aid of programming patterns, using dual pegRNA, engineering optimization to enhance pegRNA expression and strategies to add ssDNA binding domains, have improved the efficiency of editing of PE to some extent, but existing improved techniques, editing efficiency is still low and need to be explored and developed continuously.

Disclosure of Invention

In a first aspect, the present invention provides an improved guided editing system comprising a pegRNA and a fusion protein consisting of nCas9 (H840A) and M-MLV, wherein the N-terminus of nCas9 (H840A) is linked to a T5 exonuclease or Pol-N exonuclease;

the pegRNA sequentially comprises a sgRNA sequence and a PBR+RT sequence;

the amino acid sequence of nCas9 (H840A) is shown as SEQ ID NO. 1;

the amino acid sequence of the M-MLV is shown as SEQ ID NO. 2;

the amino acid sequence of the T5 exonuclease is shown as SEQ ID NO. 3;

the amino acid sequence of the Pol-N exonuclease is shown as SEQ ID NO. 4.

Furthermore, the coding gene of nCas9 (H840A) has a nucleotide sequence shown as SEQ ID NO.5, or is a complementary matched nucleotide sequence of SEQ ID NO.5, or is a nucleotide sequence with a coding amino acid sequence shown as SEQ ID NO. 1.

Furthermore, the coding gene of the M-MLV has a nucleotide sequence shown as SEQ ID NO.6, or a complementary matched nucleotide sequence of SEQ ID NO.6, or a nucleotide sequence with a coding amino acid sequence shown as SEQ ID NO. 2.

Furthermore, the coding gene of the T5 exonuclease has a nucleotide sequence shown as SEQ ID NO.7, or a complementary matched nucleotide sequence of the SEQ ID NO.7, or a nucleotide sequence with a coding amino acid sequence shown as SEQ ID NO. 3.

Furthermore, the encoding gene of the Pol-N exonuclease has a nucleotide sequence shown as SEQ ID NO.8, or a complementary paired nucleotide sequence of SEQ ID NO.8, or a nucleotide sequence with an encoding amino acid sequence shown as SEQ ID NO. 4.

In a second aspect, the invention provides the use of the guidance editing system described in any of 1) -4):

1) Editing the genome sequence of an organism or a biological cell;

2) Preparing an edited product of a genomic sequence of an organism or a biological cell;

3) Improving the editing efficiency of genome sequences of organisms or biological cells;

4) A product is prepared that increases the efficiency of editing the genomic sequence of an organism or biological cell.

The organism is a plant or an animal.

Further, the editing is base substitution, base insertion and base deletion.

In a third aspect, the present invention provides a method for editing a genomic sequence, comprising the steps of:

allowing the organism or biological cell to express the guided editing system;

the organism is a plant or an animal.

In a fourth aspect, the present invention provides a method for preparing a biological mutant, wherein the genome of an organism is edited by using the guided editing system to obtain the biological mutant;

the organism is a plant or an animal.

The invention has the following beneficial effects:

the guiding editing system is used for editing the rice genome, so that the efficiency of guiding editing is remarkably improved, and the development and application of the guiding editing system are greatly promoted.

Drawings

FIG. 1 is a schematic diagram of the operation of the optimized PE2 variant.

FIG. 2 is a schematic diagram of the vector structure of the PE2 v1-v7 variant, with NLS as the nuclear localization signal.

FIG. 3 is a graph showing the efficiency of editing in rice protoplasts of the editing system, A is the efficiency of editing 7 different PE variants at the peg-IPA1 site, and editing position is calculated from the DNA editing strand break site. B is the editing efficiency of 3 different PE2 variants at 4 rice targeting sites compared by using a rice protoplast transient transformation experiment, and the editing position is calculated from the DNA editing chain breaking site.

FIG. 4 is a gene editing of the OsAAP6 locus of the editing system in transgenic rice plants. A is a schematic diagram of partial OsAAP6 targeted gene locus and pegRNA design, wherein the underlined and bold type double represents PAM sequence, the underlined represents BamHI restriction enzyme cutting site generated after editing, and the bold type represents PBS sequence. B is the PCR/RE detection result of the aap6 mutant induced by PE2 v1 and v 2. WT/D and WT/UD bands represent the electrophoresis patterns of the wild-type plant PCR amplification products before and after BamHI cleavage, respectively. C is the target editing rate and the frequency of byproducts generated in T0 generation plants generated by PE2 v1 and v2 variant editing peg-AAP6 sites. D is the percentage of peg-AAP6 site homozygous, heterozygous and chimeric mutations in the T0 generation plants. Mo, chimera; ho, homozygote; he, hybrid. E is Hi-Tom sequencing analysis peg-AAP6 editing site representative genotype. De represents the target editing site and By represents a byproduct.

Detailed Description

The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.

The present invention discloses an improved guided editing system, which is optimized based on PE2 system (figure 1), and different 5'-3' exonuclease proteins are introduced to form different PE variants. The guide editing system comprises pegRNA and fusion protein, wherein the fusion protein is composed of nCas9 (H840A) and M-MLV, and the N end of the nCas9 (H840A) is connected with T5 exonuclease or Pol-N exonuclease;

the pegRNA sequentially comprises a sgRNA sequence and a PBR+RT sequence;

the amino acid sequence of nCas9 (H840A) is shown as SEQ ID NO. 1; the coding gene has a nucleotide sequence shown as SEQ ID NO. 5;

the amino acid sequence of the M-MLV is shown as SEQ ID NO. 2; the coding gene has a nucleotide sequence shown as SEQ ID NO. 6;

the amino acid sequence of the T5 exonuclease is shown as SEQ ID NO. 3; the coding gene has a nucleotide sequence shown as SEQ ID NO. 7;

the amino acid sequence of the Pol-N exonuclease is shown as SEQ ID NO. 4; the coding gene has a nucleotide sequence shown as SEQ ID NO. 8.

Example 1: PE2 v1-v7 vector construction

1. PE2 v1 vector construction

(1) Point mutations obtained nCas9 (H840A). Synthetic primers H840A-F (SEQ ID NO. 9) and H840A-R (SEQ ID NO. 10) were designed, and the pHUE411 plasmid was used as a template, and the point mutation was performed to obtain pHUE411-nCas9 (H840A).

(2) PE2 v1 vector was constructed (FIG. 2). On the basis of the vector pHUE411-nCas9 (H840A), mlul and Sacl are subjected to double digestion, a TGA stop codon is destroyed, an AKS intermediate fragment is connected, aflII, kpnI and SacI restriction enzyme digestion sites are introduced, and the vector pHUE411-nCas9 (H840A) -AKS is obtained. AKS amplification primers are MluI-F (SEQ ID NO. 11) and AKS-R (SEQ ID NO. 12), and the template is pHUE411 plasmid.

Then, pHUE411-nCas9 (H840A) -AKS is tangentially treated by AflII and SacI, and linker and M-MLV reverse transcriptase of 32aa are introduced into the C end of nCas9 (H840A), so as to obtain PE2 v1 vector. The primers used were AflII-MMLV-F (SEQ ID NO. 13) and SacI-MMLV-R (SEQ ID NO. 14), and the template was MMLV-MCP (commercial synthesis, nucleotide sequence shown as SEQ ID NO. 47).

2. PE2 v2-v7 vector construction

(1) PE2 v2 and v3 vector construction (FIG. 2). The XmaJI single enzyme digestion linearizes the PE2 v1 vector, adds T5 exonuclease and Pol-N exonuclease at N end of nCas9 (H840A) respectively, and obtains PE2 v2 and PE2 v3 vectors. Primers used for amplifying the T5 exonuclease are XmaJI-T5-F (SEQ ID NO. 15) and XmaJI-T5-R (SEQ ID NO. 16), and a template is T5exo (commercial synthesis, the nucleotide sequence of which is shown as SEQ ID NO. 7); primers used for amplifying Pol-N exonuclease were XmaJI-Pol-F (SEQ ID NO. 17) and XmaJI-Pol-R (SEQ ID NO. 18), and templates were: pol-N exo (commercial synthesis, nucleotide sequence shown in SEQ ID NO. 8).

(2) PE2 v4-v7 vector construction (FIG. 2). Primers AflII-MMLV-F (SEQ ID NO. 19) and KpnI-XTEN-R (SEQ ID NO. 20) were designed to amplify the 32aa linker-MMLV-NLS-T2A-MCP fragment, the template was MMLV-MCP. On the basis of pHUE411-nCas9 (H840A) -AKS, utilizing AflII and Kpnl to double-digest, introducing 32aa linker-MMLV-NLS-T2A-MCP at the C end of nCas9 (H840A) to obtain pHUE411-nCas9 (H840A) -MS2-MTMX, and finally respectively introducing T5 exonuclease and Pol-N exonuclease into pHUE411-nCas9 (H840A) -MS2-MTMX vector by Kpnl single-digest, wherein the primers for amplifying the T5 exonuclease are KpnI-T5- (SEQ ID NO. 21) and KpnI-T5-R (SEQ ID NO. 22), and the template is T5exo; the primers used for amplifying Pol-N exonuclease were KpnI-Pol-F (SEQ ID NO. 23) and KpnI-Pol-R (SEQ ID NO. 24), and the template was Pol-N exo. Amplifying the MS2-sgRNA serving as a template by using a front primer containing a targeting site and a rear primer containing a PBR+RT sequence and obtaining pegRNA, and respectively connecting the pegRNA into the vectors to obtain PE2 v4 and v6 vectors; and (3) using eMS2-sgRNA as a template, amplifying by using a front primer containing a targeting site and a rear primer containing a PBR+RT sequence to obtain pegRNA, and respectively connecting the pegRNA into the vectors to obtain PE2 v5 and v7 vectors.

Example 2: editing efficiency of targeting IPA1 gene to detect PE2 v1-v7 vector

1. Design peg-IPA1, MS2-peg-IPA1 and eMS2-peg-IPA1

(1) Restriction endonucleases BsaI-HF-V2 digested PE 2V 1, PE 2V 2 and PE 2V 3 vectors respectively, and with pHUE411 vector as template, peg-IPA1 (Table 1) and peg-IPA1-F (SEQ ID NO. 25) and peg-IPA1-R (SEQ ID NO. 26) were amplified, and peg-IPA1 was ligated into the three vectors by seamless cloning to construct IPA1-PE 2V 1, IPA1-PE 2V 2 and IPA1-PE 2V 3.

TABLE 1 sgRNA target, RT template and PBS sequence

Note that: the three bases after the sgRNA sequence are PAM sequences, and the RT templates and PBS sequences are underlined to indicate PBS sequences.

(2) Primers peg-IPA1-MS2-F (SEQ ID NO. 37) and peg-IPA1-MS2-R (SEQ ID NO. 38) are designed, the synthesized MS2-sgRNA (SEQ ID NO. 39) is used as a template for amplification to obtain MS2-peg-IPA1, and seamless cloning is respectively connected into PE2 v4 and PE2 v6 vectors to form MS2-IPA1-PE2 v4 and MS2-IPA1-PE2 v6.

(3) According to the method, eMS2-peg-IPA1 is designed, primers are peg-IPA1-eMS2-F (SEQ ID NO. 40) and peg-IPA1-eMS2-R (SEQ ID NO. 41), and synthesized eMS2-sgRNA (SEQ ID NO. 42) is used as a template, and seamless cloning is respectively connected into PE2 v5 and PE2 v7 vectors to form eMS2-IPA1-PE2 v5 and eMS2-IPA1-PE2 v7.

2. Rice protoplast transformation

Protoplasts were extracted from japonica rice Japanese sunny and were prepared in the study report of the isolation method by Zhang et al (Zhang, Y.et al A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chlorinated-related processes.2021. plant Methods 7, 30.). Taking 20 mug of the seven different plasmid vectors into a 2ml centrifuge tube, respectively adding 200ul of protoplast, adding 220ul of newly prepared PEG solution, designing three biological repetitions for each plasmid, gently reversing and mixing uniformly, and culturing at room temperature in a dark place for 20min to induce transformation; after the transformation is finished, slowly adding 880ul of the W5 solution, gently reversing and uniformly mixing, horizontally centrifuging for 3min at 250g, continuously adding 1ml of the W1 solution for resuspension, and incubating at 23 ℃ in a dark place for 48h; after the incubation, genomic DNA of the transformation product was extracted by CTAB method.

3. peg-IPA1 protoplast transformation mutation efficiency assay

The mutation sites are amplified by PCR, and the mutation efficiency of peg-IPA1 sites of different vectors is detected by second generation sequencing. Analysis of the second generation sequencing results showed that PE2 v2 and v3 vectors increased editing efficiency by 1.2-1.5 fold over PE2 v1 at peg-IPA1 site (FIG. 3A), while PE2 v4-v7 vectors were dramatically reduced in editing efficiency. The results indicate that fusing 5'-3' exonuclease at the N-terminus of PE2 nCas9 (H840A) can increase the editing efficiency of PE (fig. 3A).

Example 3: editing efficiency of targeting AAP6, PDS, OSD1 and ALS genes to detect PE2 v2 and PE2 v3 vectors

1. peg-AAP6 (FIG. 4A), peg-PDS, peg-OSD1, peg-ALS (Table 1) were designed. Respectively inserting into PE2 v1, PE2 v2 and PE2 v3 vectors according to the method, transferring into rice protoplast, extracting DNA, amplifying corresponding target spots by PCR, and detecting mutation efficiency by second generation sequencing.

2. And analyzing editing efficiency of different targets. Analytical sequencing results showed that at four targets the editing efficiency of the PE2 v2 variants was increased by 1.48-fold (peg-AAP 6), 4.9-fold (peg-OSD 1), 1.3-fold (peg-OSD 1) and 2.92-fold (peg-ALS), respectively, whereas for the PE2 v3 variants the editing efficiency was increased by 5.4-fold and 1.35-fold at only two targets of peg-PDS and peg-ALS. Experimental results show that the T5 or Pol-N exonuclease introduced into the N end of PE2 nCas9 (H840A) can improve the editing efficiency of PE, and the T5 exonuclease has better effect compared with Pol-N exonuclease.

Example 4: analysis of mutation efficiency in genetic transformation of rice

1. Culturing with japonica rice Nipponbare as material to obtain rice callus. The editing vectors AAP6-PE2 v1 and AAP6-PE2 v2 of peg-AAP6 site (FIG. 4A) were transferred into EHA105 Agrobacterium strain by liquid nitrogen freeze-thawing transformation method, and transferred into rice callus by means of Agrobacterium Agrobacterium tumefaciens. Rice callus culture and transformation methods are described in the previous report by Hiei et al (Hiei, Y. & Komari, T.Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed, 2008.Nature protocols.3, 824-834). Screening and culturing embryogenic callus containing agrobacterium on a culture medium containing 50mg/L hygromycin to obtain resistant callus, and continuing culturing to obtain regenerated T0 generation transgenic rice.

2. PCR/RE detection (FIG. 4B). AAP6 Gene PCR amplification primers 1st-AAP6-F (SEQ ID NO. 43) and 1st-AAP6-F (SEQ ID NO. 44) were designed. Cutting 2-3cm of regenerated T0 generation rice plant leaves, extracting leaf DNA by a CTAB method, and amplifying an AAP6 gene fragment containing a mutation site by using the primers to about 754bp. 5. Mu.l of the amplified product was aspirated, 0.5. Mu.l of BamHI enzyme was added, 2. Mu.l of 10X BamHI fast digest buffer was added, the volume was set to 20. Mu.l, incubated at 37℃for 2 hours, and the result of cleavage was observed under UV conditions by electrophoresis on a 2% agarose gel. If the band is completely cut, it is demonstrated that the homozygous mutant is made; if the band portion is cut, the mutant may be a heterozygous or chimeric mutation.

The results of the digestion show (FIG. 4C) that 17 out of 43T 0 transgenic plants regenerated from PE2 v1 can be digested with BamHI enzyme with a mutation efficiency of 39.5%. Wherein, the PCR fragment from the T0-10 mutant is completely cut, and the possibility of a homozygous mutant is proved; such as T0-9, 19 and 40, etc.; the PCR fragment was partially cleaved, and a chimeric mutant was possible. For the PE2 v2 variant, after BamHI digestion, 17 mutants were detected in 36 regenerated T0 generation plants with an editing efficiency of 47.22% in which the PCR fragments of 5 plants were completely digested.

3. High throughput sequencing analysis to determine mutation type

Primers Hitom-AAP6-F (SEQ ID NO. 45) and Hitom-AAP6-R (SEQ ID NO. 46) were designed, the mutant fragment was amplified, and Hi-Tom high throughput sequencing was performed to determine the mutation type.

Analysis of the sequencing results showed that of the 17 mutants of PE2 v1, the homozygous mutation was 5.88%, the heterozygous mutation was 58.83%, the chimera was 35.29%, and by-product production was observed in the three mutant lines (fig. 4d, e). For the PE2 v2 variant, the homozygous mutation rate was 29.41%, the heterozygous mutation rate was 64.71%, the chimera was 5.88%, and the by-product was contained in only one strain. Experimental results show that at peg-AAP6 site, PE2 v2 editing efficiency is improved by 1.34 times compared with PE2 v1, homozygous mutation rate is increased by 5 times, and chimeric mutation rate is reduced.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (9)

1. An improved guided editing system comprising pegRNA and a fusion protein consisting of nCas9 (H840A) and M-MLV, wherein the N-terminus of nCas9 (H840A) is linked to a T5 exonuclease or Pol-N exonuclease;

the pegRNA sequentially comprises a sgRNA sequence and a PBR+RT sequence;

the amino acid sequence of nCas9 (H840A) is shown as SEQ ID NO. 1;

the amino acid sequence of the M-MLV is shown as SEQ ID NO. 2;

the amino acid sequence of the T5 exonuclease is shown as SEQ ID NO. 3;

the amino acid sequence of the Pol-N exonuclease is shown as SEQ ID NO. 4.

2. The guidance editing system of claim 1, wherein the encoding gene of nCas9 (H840A) has the nucleotide sequence shown in SEQ ID No.5, or is the complementary paired nucleotide sequence of SEQ ID No.5, or is the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1.

3. The guidance editing system of claim 1, wherein the coding gene of M-MLV has a nucleotide sequence shown as SEQ ID No.6, or a complementary pair of nucleotide sequences of SEQ ID No.6, or a nucleotide sequence encoding an amino acid sequence shown as SEQ ID No. 2.

4. The guidance editing system of claim 1, wherein the coding gene of the T5 exonuclease has a nucleotide sequence shown as SEQ ID No.7, or a complementary pair of nucleotide sequences of SEQ ID No.7, or a nucleotide sequence encoding an amino acid sequence shown as SEQ ID No. 3.

5. The guidance editing system of claim 1, wherein the Pol-N exonuclease-encoding gene has a nucleotide sequence shown as SEQ ID No.8, or a complementary pair of nucleotide sequences of SEQ ID No.8, or a nucleotide sequence encoding an amino acid sequence shown as SEQ ID No. 4.

6. The use of the guided editing system of claim 1 in any one of 1) -4):

1) Editing the genome sequence of an organism or a biological cell;

2) Preparing an edited product of a genomic sequence of an organism or a biological cell;

3) Improving the editing efficiency of genome sequences of organisms or biological cells;

4) A product is prepared that increases the efficiency of editing the genomic sequence of an organism or biological cell.

The organism is a plant or an animal.

7. The use of claim 6, wherein the editing is base substitution, base insertion and base deletion.

8. A method for editing a genomic sequence, comprising the steps of:

causing an organism or biological cell to express the guided editing system of claim 1;

the organism is a plant or an animal.

9. A method for producing a biological mutant, comprising editing a genome of an organism using the guidance editing system according to claim 1, thereby obtaining the biological mutant;

the organism is a plant or an animal.

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