CN116064512A - An Improved Guidance Editing System and Its Application - Google Patents

Abstract

The invention relates to the field of genome editing, in particular to an improved guided editing system and its application. The guide editing system includes pegRNA and fusion protein, the fusion protein is composed of nCas9 (H840A) and M-MLV, wherein, the N-terminus of nCas9 (H840A) is connected with T5 exonuclease or Pol-N exonuclease ; The pegRNA is sequentially composed of sgRNA and PBR+RT sequence. The editing efficiency of the guidance editing system of the present invention is further improved on the basis of the prior art, which greatly promotes the development and application of the guidance editing system.

Description

An Improved Guidance Editing System and Its Application

technical field

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

Background technique

Clustered regularly interspaced short palindromic repeats associated protein (CRISPR/Cas) has almost replaced all genome editing technologies due to its simplicity, speed, and efficiency, and has been widely used in animal in plant research. The CRISPR/Cas9 system is mainly composed of the Cas9 endonuclease protein and single-stranded guide RNA (single guide RNA, sgRNA). Under the guidance of the sgRNA, the Cas9 protein recognizes a specific target, and the adjacent motif (Protospacer Adjacent motif (PAM) cuts to generate a DNA double-strand break (DNA double-strandbreak, DSB), which triggers the body's self-repair mechanism and realizes DNA site-directed mutation. The common DSBs repair pathways in cells are non-homologous end-joining (Non-homologous end-joining, NHEJ) and homologous recombination (Homologous recombination, HR). HR is a precise repair pathway that requires the participation of donor templates and can achieve precise site-directed mutations. However, HR usually has a complex vector design, mainly occurs in the G2 phase and S phase of cell division, is limited by the editing period, and the editing efficiency is low, so it is difficult to be widely used. NHEJ is an error-prone repair pathway, which usually can only introduce random insertion/deletion (insertion and deletion, indels) of a small range of bases, and the variety of mutation types is difficult to control. Genome editing technologies derived from the CRISPR/Cas system include base editing (BE) and prime editing (PE).

Conventional BE includes cytosine base editor (CBE) and adenine base editor (ABE), mainly composed of cytosine deaminase/adenine deaminase, nCas9 (nickase Cas9) and sgRNA. Under the guidance of sgRNA, the fusion protein of nCas9 and deaminase binds to the target, deaminates specific bases, and then generates base-specific substitutions through DNA damage repair. Cytosine deaminase recognizes deaminated cytosine (C) to form uracil (U), and then converts U to T through DNA replication or repair, and finally realizes the substitution of C-G to A-T base pairs. The most widely used cytosine deaminase is rat-derived cytidine deaminase (rAPOBEC1). Adenine deaminase recognizes adenine (A) and deaminates to form inosine (I). Since the chemical structure of I is similar to G, it is mistakenly recognized as G during DNA repair, and T in the repair complementary chain is C , and then realize the conversion from A-T to G-C. The commonly used adenine deaminase is a heterodimer composed of optimized Escherichia coli tRNA adenine deaminase (ecTadA*) and wild-type ecTadA. BE can achieve precise single-base mutations at target sites, but currently the two base editors can only achieve C-T and A-G conversions, not transversions, and are easily limited by the editing window, with limited editing types and scopes. Whole-genome sequencing found that BE also caused a certain degree of off-target mutations.

The invention and application of PE is an innovative breakthrough in genome editing technology. PE1 is composed of two parts: a fusion protein formed by nCas9 (H840A) and reverse transcriptase M-MLV, and a pegRNA formed by connecting a primer binding site (PBS), a reverse transcription template sequence (RT template) and sgRNA. The fusion protein composed of nCas9(H840A) and M-MLV cuts single-stranded DNA under the guidance of pegRNA, and the PBS at the 3' end of pegRNA binds to the sequence at the cutting breakpoint through base pairing. Under the action of M-MLV, along The RT template is reverse-transcribed to synthesize 3' cohesive ends containing the mutation of the target gene. At this time, the 3' end containing the target edit and the 5' end containing the unedited sequence will be formed at the break at the same time. Under the action of the 5'-3' exonuclease in the cell, the 5' end will be cut and the 3' end will be connected, and then Realize precise gene site-directed mutation. On the basis of PE1, PE2 is engineered to optimize the M-MLV protein, which increases the editing efficiency compared to PE1. On the basis of PE2 pegRNA, PE3 additionally adds sgRNA to target the complementary DNA strand of the pegRNA-guided cleavage strand and regulate the intracellular DNA repair mechanism. However, studies have shown that PE3 has no editing effect on multiple targets. PE can realize almost any type of small fragment gene mutation, including 12 different types of base substitutions (transitions and transversions), deletion and insertion of small fragments, etc., and does not require donor templates and double-strand DNA breaks.

Although PE is powerful, its application scope is greatly limited due to the low editing efficiency. Some substantive efforts, including strategies such as designing pegRNAs with the help of programmed patterns, using double pegRNAs, engineering optimization to enhance pegRNA expression, and adding ssDNA binding domains, have improved the editing efficiency of PE to a certain extent, but existing improved technologies, Editing efficiency is still low, and needs to be continuously explored and developed.

Contents of the invention

In the first aspect, the present invention provides an improved guide editing system, including pegRNA and fusion protein, the fusion protein is a fusion protein composed of nCas9(H840A) and M-MLV, wherein the N-terminus of nCas9(H840A) is connected With T5 exonuclease or Pol-N exonuclease;

The pegRNA consists of sgRNA and PBR+RT sequence in turn;

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

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

The amino acid sequence of described T5 exonuclease is as shown in SEQ ID NO.3;

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

Further, the coding gene of the nCas9 (H840A) has the nucleotide sequence shown in SEQ ID NO.5, or the nucleotide sequence complementary to SEQ ID NO.5, or the encoded amino acid sequence such as SEQ ID NO .1 the nucleotide sequence.

Further, the coding gene of the M-MLV has the nucleotide sequence shown in SEQ ID NO.6, or the nucleotide sequence complementary to SEQ ID NO.6, or the encoded amino acid sequence such as SEQ ID NO.2 the nucleotide sequence.

Further, the coding gene of the T5 exonuclease has the nucleotide sequence shown in SEQ ID NO.7, or the nucleotide sequence complementary to SEQ ID NO.7, or the encoded amino acid sequence such as SEQ ID Nucleotide sequence of NO.3.

Further, the coding gene of the Pol-N exonuclease has the nucleotide sequence shown in SEQ ID NO.8, or the nucleotide sequence complementary to SEQ ID NO.8, or the coding amino acid sequence such as The nucleotide sequence of SEQ ID NO.4.

In a second aspect, the present invention provides the application of the guide editing system in any of 1)-4):

1) Editing of genome sequences of organisms or biological cells;

2) Preparation of edited products of organism or biological cell genome sequence;

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

4) Preparation of products that improve the editing efficiency of genome sequences of organisms or biological cells.

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 genome sequence, comprising the following steps:

causing the organism or biological cell to express the guide editing system;

The organism is a plant or an animal.

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

The organism is a plant or an animal.

The present invention has following beneficial effect:

Applying the guided editing system of the present invention to edit the rice genome significantly improves the efficiency of guided editing, and greatly promotes the development and application of the guided editing system.

Description of drawings

Figure 1 is a schematic diagram of the optimization of PE2 variants.

Figure 2 is a schematic diagram of the vector structure of PE2 v1-v7 variants, and NLS is the nuclear localization signal.

Figure 3 is the verification of the editing efficiency of the editing system in rice protoplasts. A is the editing efficiency of seven different PE variants at the peg-IPA1 site, and the editing position is calculated from the DNA editing strand break site. B is the comparison of editing efficiency of 3 different PE2 variants at 4 rice targeting sites using rice protoplast transient transformation experiments, and the editing position is calculated from the DNA editing strand break site.

Fig. 4 shows the gene editing of the OsAAP6 locus in rice transgenic plants by the editing system. A is a schematic diagram of part of the OsAAP6 targeting gene locus and pegRNA design. The underline and bold double indicate the PAM sequence, the underline indicates the BamHI restriction enzyme site generated after editing, and the bold indicates the PBS sequence. B is the PCR/RE detection result of aap6 mutant induced by PE2 v1 and v2. The WT/D and WT/UD bands represent the electrophoresis of the PCR amplification products of wild-type plants before and after BamHI digestion. C is the targeted editing rate and by-product frequency in the T0 generation plants produced by PE2 v1 and v2 variants editing the peg-AAP6 site. D is the percentage of homozygous mutation, heterozygous mutation and chimeric mutation at peg-AAP6 site in T0 plants. Mo, chimera; Ho, homozygote; He, heterozygote. E is the representative genotype of peg-AAP6 editing site analyzed by Hi-Tom sequencing. De indicates the target editing site and By indicates the byproduct.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but should not be construed as a limitation of the present invention. Unless otherwise specified, 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, unless otherwise specified, can be obtained from commercial sources.

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

The pegRNA consists of sgRNA and PBR+RT sequence in turn;

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

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

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

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

Embodiment 1: PE2 v1-v7 vector construction

1. PE2 v1 vector construction

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

(2) Construction of PE2 v1 vector (Figure 2). On the basis of the above-mentioned vector pHUE411-nCas9 (H840A), Mlul and Sacl were double-digested, the TGA stop codon was destroyed, the AKS middle fragment was connected, and AflII, KpnI and SacI restriction enzyme sites were introduced to obtain the vector pHUE411-nCas9( H840A) - AKS. AKS amplification primers are MluI-F (SEQ ID NO.11) and AKS-R (SEQ ID NO.12), and the template is pHUE411 plasmid.

Then, AflII and SacI were digested to linearize pHUE411-nCas9(H840A)-AKS, and a 32aa linker and M-MLV reverse transcriptase were introduced into the C-terminus of nCas9 (H840A) to obtain a PE2 v1 vector. The primers used are AflII-MMLV-F (SEQ ID NO.13) and SacI-MMLV-R (SEQ ID NO.14), and the template is MMLV-MCP (commercially synthesized, the nucleotide sequence is shown in SEQ ID NO.47).

2. PE2 v2-v7 carrier construction

(1) PE2 v2 and v3 vector construction (Figure 2). XmaJI single-enzyme digestion linearized PE2 v1 vector, and added T5 exonuclease and Pol-N exonuclease to the N-terminus of nCas9(H840A), respectively, to obtain PE2 v2 and PE2 v3 vectors. Primers used to amplify T5 exonuclease are XmaJI-T5-F (SEQ ID NO.15) and XmaJI-T5-R (SEQ ID NO.16), and template is T5exo (commercial synthesis, nucleotide sequence such as SEQ ID shown in NO.7); the primers used to amplify Pol-N exonuclease are XmaJI-Pol-F (SEQ ID NO.17) and XmaJI-Pol-R (SEQ ID NO.18), and the template is: Pol- N exo (commercial synthesis, nucleotide sequence as shown in SEQ ID NO.8).

(2) PE2 v4-v7 vector construction (Figure 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, and the template was MMLV-MCP. On the basis of pHUE411-nCas9(H840A)-AKS, use AflII and Kpnl double enzyme digestion, introduce 32aa linker-MMLV-NLS-T2A-MCP at the C-terminus of nCas9(H840A), and obtain pHUE411-nCas9(H840A)-MS2 -MTMX, finally through Kpnl single enzyme digestion, respectively introduce T5 exonuclease and Pol-N exonuclease into the pHUE411-nCas9(H840A)-MS2-MTMX vector, and the primers used to amplify T5 exonuclease are primers Be KpnI-T5-(SEQ ID NO.21) and KpnI-T5-R (SEQ ID NO.22), template is T5exo; The primer used for amplifying Pol-N exonuclease is KpnI-Pol-F (SEQ ID NO. .23) and KpnI-Pol-R (SEQ ID NO.24), the template is Pol-N exo. Using MS2-sgRNA as a template, amplify the pegRNA with the front primer containing the targeting site and the back primer containing the PBR+RT sequence and respectively, and connect them into the above vectors respectively to obtain PE2v4 and v6 vectors; use eMS2-sgRNA as the The template is amplified with the front primer containing the targeting site and the back primer containing the PBR+RT sequence to obtain pegRNA, which are respectively ligated into the above vectors to obtain PE2 v5 and v7 vectors.

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

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

(1) Restriction endonuclease BsaI-HF-V2 digested PE2 v1, PE2 v2 and PE2 v3 vectors respectively, using the pHUE411 vector as a template to amplify peg-IPA1 (Table 1), and the primer was peg-IPA1-F( SEQ ID NO.25) and peg-IPA1-R (SEQ ID NO.26), peg-IPA1 was connected to the above three vectors through seamless cloning to form IPA1-PE2 v1, IPA1-PE2v2, IPA1-PE2 v3.

Table 1 sgRNA target, RT template and PBS sequence

Note: The last three bases of the sgRNA sequence are the PAM sequence, and the RT template and PBS sequence are underlined to indicate the PBS sequence.

(2) Design primers peg-IPA1-MS2-F (SEQ ID NO.37) and peg-IPA1-MS2-R (SEQ ID NO.38), and use the synthesized MS2-sgRNA (SEQ ID NO.39) as a template to amplify MS2-peg-IPA1 was added, and seamlessly cloned into PE2 v4 and PE2 v6 vectors to form MS2-IPA1-PE2 v4 and MS2-IPA1-PE2 v6.

(3) According to the above method, design eMS2-peg-IPA1, the 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) was used as a template, and seamlessly cloned into PE2 v5 and PE2 v7 vectors to form eMS2-IPA1-PE2 v5 and eMS2-IPA1-PE2 v7.

2. Transformation of rice protoplasts

The protoplasts were extracted from japonica rice Nipponbare, and the protoplasts were prepared according to Zhang et al. A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes.2021. Plant Methods 7 ,30.) research reports. Take 20μg of the above seven different plasmid vectors into a 2ml centrifuge tube, add 200ul protoplasts respectively, and then add 220ul newly prepared PEG solution, design three biological replicates for each plasmid, gently invert and mix, and incubate at room temperature in the dark 20min to induce transformation; after transformation, slowly add 880ul W5 solution, mix gently by inversion, centrifuge horizontally at 250g for 3min, continue to add 1ml W1 solution to resuspend, and incubate in the dark at 23°C for 48h; after incubation, extract the transformation product by CTAB method Genomic DNA.

3. Detection of mutation efficiency of peg-IPA1 protoplast transformation

The mutation site was amplified by PCR, and the mutation efficiency of the peg-IPA1 site of different vectors was detected by next-generation sequencing. Analysis of next-generation sequencing results showed that at the peg-IPA1 site, the editing efficiency of PE2 v2 and v3 vectors increased by 1.2-1.5 times compared to PE2 v1 (Figure 3A), while the editing efficiency of PE2 v4-v7 vectors dropped sharply. The results showed that fusion of 5'-3' exonuclease at the N-terminus of PE2 nCas9(H840A) increased the editing efficiency of PE (Fig. 3A).

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

1. Design peg-AAP6 (Figure 4A), peg-PDS, peg-OSD1, peg-ALS (Table 1). Insert PE2 v1, PE2 v2 and PE2 v3 vectors according to the above method, and transform them into rice protoplasts, extract DNA, amplify the corresponding target sites by PCR, and detect the mutation efficiency by next-generation sequencing.

Second, analyze the editing efficiency of different targets. Analysis of the sequencing results showed (Fig. 3B) that at the four targets, the editing efficiency of the PE2 v2 variant was increased by 1.48 times (peg-AAP6), 4.9 times (peg-OSD1), 1.3 times (peg-OSD1) and 2.92-fold (peg-ALS), while for the PE2 v3 variant, the editing efficiency was only increased by 5.4-fold and 1.35-fold at the two targets of peg-PDS and peg-ALS. The experimental results showed that the introduction of T5 or Pol-N exonuclease at the N-terminus of PE2 nCas9(H840A) could improve the editing efficiency of PE, and T5 exonuclease had a better effect than Pol-N exonuclease. better.

Embodiment 4: Rice genetic transformation analysis mutation efficiency

1. The rice callus was obtained by culturing the japonica rice Nipponbare. The editing vectors AAP6-PE2 v1 and AAP6-PE2 v2 of the peg-AAP6 site (Figure 4A) were transformed into the EHA105 Agrobacterium strain by liquid nitrogen freeze-thaw transformation method, and then transformed into the rice callus with the help of Agrobacterium tumefaciens. Rice callus culture and transformation methods refer to previous reports 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). The embryogenic callus containing Agrobacterium was screened and cultured on a medium containing 50 mg/L hygromycin to obtain resistant callus, and the culture was continued 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. Cut out 2-3cm leaves of regenerated T0 generation rice plants, extract leaf DNA by CTAB method, and use the above primers to amplify the AAP6 gene fragment containing the mutation site, about 754bp. Pipette 5 μl of the amplified product, add 0.5 μl of BamHI enzyme, add 2 μl of 10×BamHI fast digest buffer, make the volume to 20 μl, incubate at 37°C for 2 hours, run 2% agarose gel electrophoresis, and observe the digestion results under ultraviolet conditions. If the band is cut completely, it proves to be a homozygous mutant; if the band is partially cut, the mutant may be heterozygous or chimeric.

The enzyme digestion results showed (Fig. 4C), among the 43 T0 transgenic plants regenerated from PE2 v1, 17 plants could be cut by BamHI enzyme, and the mutation efficiency was 39.5%. Among them, if the PCR fragment from the T0-10 mutant strain is completely cut, it may be a homozygous mutant; such as T0-9, 19 and 40, etc.; if the PCR fragment is partially cut, it may be a chimeric mutant. For the PE2v2 variant, after BamHI digestion, 17 mutants were detected in 36 regenerated T0 plants, with an editing efficiency of 47.22%, and the PCR fragments of 5 plants were completely digested.

3. High-throughput sequencing analysis to determine the mutation type

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

Analysis and sequencing results showed (Fig. 4D, E), among the 17 mutants of PE2 v1, the homozygous mutation was 5.88%, the heterozygous mutation was 58.83%, and the chimera was 35.29%, and it was observed in three mutant lines By-products are produced. For the PE2 v2 variant, the homozygous mutation rate was 29.41%, the heterozygous mutation rate was 64.71%, and the chimera was 5.88%, and only one line contained the byproduct. The experimental results showed that at the peg-AAP6 site, the editing efficiency of PE2 v2 was 1.34 times higher than that of PE2 v1, the homozygous mutation rate was increased by 5 times, and the chimeric mutation rate was reduced.

While preferred embodiments of the invention have been described, additional changes and modifications to these embodiments can be made by those skilled in the art once the basic inventive concept is appreciated. Therefore, it is intended that the appended claims be construed to cover the preferred embodiment as well as all changes and modifications which fall within the scope of the invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims (9)

1. An improved guided editing system, characterized in that it comprises pegRNA and fusion protein, and the fusion protein is a fusion protein composed of nCas9 (H840A) and M-MLV, wherein the N-terminus of nCas9 (H840A) is connected with T5 exonuclease or Pol-N exonuclease; The pegRNA consists of sgRNA and PBR+RT sequence in turn; The amino acid sequence of the nCas9 (H840A) is shown in SEQ ID NO.1; The amino acid sequence of the M-MLV is shown in SEQ ID NO.2; The amino acid sequence of described T5 exonuclease is as shown in SEQ ID NO.3; The amino acid sequence of the Pol-N exonuclease is shown in SEQ ID NO.4. 2. guide editing system according to claim 1, is characterized in that, the coding gene of described nCas9 (H840A) has the nucleotide sequence shown in SEQ ID NO.5, or is the complementary pairing of SEQ ID NO.5 A nucleotide sequence, or a nucleotide sequence encoding an amino acid sequence such as SEQ ID NO.1. 3. guide editing system according to claim 1, is characterized in that, the coding gene of described M-MLV has the nucleotide sequence shown in SEQID NO.6, or is the nucleoside of SEQ ID NO.6 complementary pairing An acid sequence, or a nucleotide sequence encoding an amino acid sequence such as SEQ ID NO.2. 4. guide editing system according to claim 1, is characterized in that, the coding gene of described T5 exonuclease has the nucleotide sequence shown in SEQ ID NO.7, or is the complementary pairing of SEQ ID NO.7 A nucleotide sequence, or a nucleotide sequence encoding an amino acid sequence such as SEQ ID NO.3. 5. guide editing system according to claim 1, is characterized in that, the coding gene of described Pol-N exonuclease has the nucleotide sequence shown in SEQ ID NO.8, or is SEQ ID NO.8 A complementary paired nucleotide sequence, or a nucleotide sequence encoding an amino acid sequence such as SEQ ID NO.4. 6. the described guidance editing system of claim 1 in 1)-4) any application in: 1) Editing of genome sequences of organisms or biological cells; 2) Preparation of edited products of organism or biological cell genome sequence; 3) Improve the editing efficiency of genome sequences of organisms or biological cells; 4) Preparation of products that improve the editing efficiency of genome sequences of organisms or biological cells. The organism is a plant or an animal. 7. The application according to claim 6, wherein the editing is base substitution, base insertion and base deletion. 8. A method for editing a genome sequence, comprising the steps of: Making organisms or biological cells express the guide editing system described in claim 1; The organism is a plant or an animal. 9. A method for preparing a biological mutant, characterized in that, using the guided editing system according to claim 1 to edit the genome of an organism to obtain a biological mutant; The organism is a plant or an animal.

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