Phase-separating nicking enzyme-mediated prime editors and editing systems and applications thereof

Abstract

This invention discloses a phased-cutting enzyme-mediated leader editor and editing system, and their applications, belonging to the field of biotechnology. This editor integrates Cas9-D10A and PE7max, and is composed of a Cas9-H840A mutant, MMLV reverse transcriptase, LA protein, Cas9-D10A mutant, and pegRNA. By synergistically utilizing the activities of the two Cas9 cutting enzymes, it generates spatially symmetrical and temporally phased DNA cuts at the target site, thereby accelerating the repair of edited products. Simultaneously, key Cas9 mutation sites are screened through protein language optimization to further optimize the editor. Results show that this editor significantly improves editing efficiency, and the optimized editor further enhances the editing efficiency of endogenous sites without significantly increasing insertions or deletions, while maintaining editing specificity for scaffold byproducts. This solves the problems of insufficient PE editing efficiency and high indel ratio in existing PE editing methods.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a symmetric nicking enzyme-mediated leader editor and editing system and its applications. Background Technology

[0002] The prime editor (PE) is one of the most advanced second-generation CRISPR / Cas9 genome editing technologies, capable of performing any type of single-base substitution as well as the insertion and deletion of short DNA fragments. The prime editor consists of a Cas9n (Cas9 nickase)-H840A mutant, MMLV reverse transcriptase, and pegRNA. Cas9n mediates the unwinding of double-stranded DNA and the cleavage of the non-target strand, inducing the generation of binding primers. Subsequently, the PBS (prime binding sequence) in the pegRNA binds to the primers, and the reverse transcriptase guides the generation of reverse transcription products. Finally, the target mutation is inserted through the DNA repair mechanism of eukaryotic cells. Currently, PE has evolved from the initial PE2 / 3 to the existing PE6 / 7 versions. Compared to PE2, PE3 introduced a gRNA that guides the cutting of the target strand, which can improve the editing efficiency. PE4 / 5 introduced the negative regulator MLH1dn, which inhibits cell MMR (Mismatch repair), to further optimize the editing efficiency of the existing PE. PE2MAX replaced MMLV with a high-efficiency reverse transcriptase truncation and further optimized PE by optimizing the nuclear localization signal and Cas9 cleavage activity. PE7max introduced the LA protein based on PE2MAX, which further optimized PE (Yan J, Oyler-Castrillo P, Ravisankar P, et al. Improving prime editing with an endogenous small RNA-binding protein[J].Nature, 2024, 628(8008):34.DOI:10.1038 / s41586-024-07259-6.). Although the editing efficiency of PE has been significantly improved, there is still room for optimization in terms of both editing efficiency and the proportion of indel products. Researchers also reduced the proportion of indel products by inducing Cas9 cut site relaxation mutations that degrade the cut ends, but the editing efficiency was low (Chauhan VP, Sharp PA, Langer R. Engineered prime editors with minimal genomic errors[J]. Nature, 2024. DOI:10.1101 / 2024.08.02.606370.).

[0003] There is still room for improvement in the editing efficiency of PE. Repeated binding of gRNA and target strand can increase the indel ratio, which is due to the unclear repair mechanism of PE in eukaryotic cells.

[0004] Therefore, developing a new PE editor that enhances PE editing efficiency and reduces indel is of great significance. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a new PE editor, PACE ( P h a sed C as9 nickase e PACE-PE (pegRNA) is an editor that integrates a heterologous nickase platform of Cas9n-D10A and Cas9n-H840A guided by a single single guide RNA. The PACE-PE architecture further expands this capability by integrating Cas9n-D10A with PE7max, achieving efficient and scalable lead editing and significantly reducing byproducts. PACE-PE consists of a Cas9n-H840A mutant, MMLV reverse transcriptase, LA protein, a Cas9n-D10A mutant, and pegRNA. Its basic principle is to synergistically generate directional double-stranded cuts under pegRNA guidance through the nickase activities of Cas9n-D10A and Cas9n-H840A, thereby accelerating the repair of PE-edited products and improving editing efficiency.

[0006] On one hand, this invention provides a symmetrical cleavage enzyme-mediated leader editor, which integrates a Cas9 cleavage enzyme 1 and Cas9 cleavage enzyme 2 platform guided by a single unidirectional guide RNA, comprising two different Cas9 cleavage enzymes, MMLV reverse transcriptase, LA protein, and pegRNA. Cas9 cleavage enzyme 1 is used to cleave the non-target strand, while Cas9 cleavage enzyme 2 is used to cleave the target strand; the cuts made by both are symmetrical. Because only one gRNA is used, the two cleavage enzymes cannot bind to the target site simultaneously, resulting in symmetrical cuts due to asynchronous cleavage by the two cleavage enzymes.

[0007] Specifically, the Cas9 nicking enzyme includes Cas9 nicking enzyme 1 and Cas9 nicking enzyme 2, wherein Cas9 nicking enzyme 1 is selected from any one or more of the following:

[0008] (i) The nickase 1 is a Cas9n-H840A mutant, whose amino acid sequence is mutated at amino acid position 840 compared with the amino acid sequence of wild-type Cas9. The amino acid sequence of the Cas9n-H840A mutant is shown in SEQ ID NO: 1.

[0009] (ii) The nickase 1 is a Cas9n-H840A-S55E-I122D-I170N-D947G mutant, whose amino acid sequence has mutations at amino acid positions 840, 55, 122, 170 and / or 947 compared with the amino acid sequence of wild-type Cas9. The amino acid sequence of the Cas9n-H840A-S55E-I122D-I170N-D947G mutant is shown in SEQ ID NO: 5.

[0010] The Cas9 nickase 2 is selected from any one or more of the following:

[0011] (a) The nickase 2 is a Cas9n-D10A mutant, whose amino acid sequence is mutated at the 10th amino acid position compared with the amino acid sequence of wild-type Cas9. The amino acid sequence of the Cas9-D10A mutant is as shown in SEQ ID NO: 4.

[0012] (b) The nickase 2 is Cas9n-D10A-N803P-D861S, whose amino acid sequence has mutations at amino acid positions 10, 803 and / or 861 compared with the amino acid sequence of wild-type Cas9. The amino acid sequence of the Cas9n-D10A-N803P-D861S mutant is as shown in SEQ ID NO: 6.

[0013] In this invention, amino acid residues can be represented by a single letter or by three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamic acid (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), and arginine (Arg, R).

[0014] As used herein, the term "H840A" indicates that amino acid H at position 840 is replaced by amino acid A, and "Cas9n-H840A" indicates that amino acid H at position 840 of the Cas9 protein is replaced by amino acid A. For example, "Cas9n-H840A-S55E-I122D-I170N-D947G" indicates that amino acid H at position 840 of the Cas9 protein is replaced by amino acid A, amino acid S at position 55 is replaced by amino acid E, amino acid I at position 122 is replaced by amino acid D, amino acid I at position 170 is replaced by amino acid N, and amino acid D at position 947 is replaced by amino acid G, and so on. The mutant site represents a mutation relative to the wild-type amino acid sequence.

[0015] Specifically, the amino acid sequence of the MMLV reverse transcriptase is shown in SEQ ID NO:2, and the amino acid sequence of the LA protein is shown in SEQ ID NO:3.

[0016] On one hand, the present invention provides a method for constructing the aforementioned pilot editor, comprising the following steps:

[0017] 1) Construct editors PE7max and Cas9 nickase 2, respectively, containing Cas9 nickase 1;

[0018] 2) gRNA was constructed by enzyme digestion and ligation, and then pegRNA was constructed by PCR and homologous recombination;

[0019] 3) Mix PE7max with pegRNA and add Cas9 cleavage enzyme 2 to obtain PACE-PE editor.

[0020] Specifically, the enzyme digestion and ligation system for constructing gRNA includes EC3IL enzyme, T4 ligase, T4 ligase buffer and BSA, wherein PE7max:pegRNA:Cas9 cleavage enzyme 2 = (2-6):(1-2):(1-1.5).

[0021] On one hand, the present invention provides a symmetric nicking enzyme-mediated pilot editing system, comprising the aforementioned pilot editor.

[0022] On one hand, the present invention provides a gene editing method, which is not for disease diagnosis and treatment purposes. The method involves transfecting cells with the aforementioned lead editor or lead editing system, adding Opti medium and PEI during transfection to achieve a DNA:PEI mass ratio of 1:2 to 10, and collecting the cell pellet after transfection to obtain gene-edited cells. Preferably, the DNA:PEI mass ratio is 1:3 to 5. Preferably, the gene editing is gene editing in eukaryotic cells, such as HEK293T cells.

[0023] Specifically, prior to transfection, the cells were prepared in 24-well plates at a ratio of 6 × 10⁻⁶.4 -10×10 4 The cells are seeded at a specific density and cultured until the cell density reaches 60%-80%. Transfection is then performed, with medium changes required 6 hours after transfection. The cell pellet is collected 70-80 hours after transfection. Preferably, the concentration is 7 × 10⁻⁶ cells / day. 4 -8×10 4 The cells were plated at a high density and the cell pellet was collected 70-75 hours after transfection.

[0024] On the one hand, the present invention provides the application of the aforementioned leader editor or the aforementioned leader editing system in improving the efficiency of genome leader editing.

[0025] On the other hand, the present invention provides the application of the aforementioned leader editor or the aforementioned leader editing system in reducing the proportion of insertion and deletion byproducts during genome leader editing.

[0026] Compared with existing technologies, this invention has the following advantages: The PACE-PE editor constructed in this invention integrates Cas9n-D10A and PE7max, synergistically generating controllable double-stranded cuts through the activities of two Cas9 nickases, significantly improving the efficiency of leader editing. At multiple sites, the editing efficiency is increased from 1.9-22.4% to 14.6-68.5%, without significantly increasing insertion / deletion byproducts. The enPACE-PE obtained through protein language optimization further improves editing efficiency at multiple endogenous sites while maintaining high editing specificity for insertions / deletions and scaffold byproducts. This invention overcomes the technical bottlenecks of existing PE editors, such as unclear repair mechanisms and high indel ratios due to repeated gRNA binding. It achieves efficient editing without puromycin screening, and its editor architecture is scalable. It provides a novel, efficient, specific, and low-byproduct tool for genome editing, possessing significant practical value in biotechnology research and gene editing applications. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the working principle and target verification of PACE-PE. In the diagram, a represents the composition and working principle of the PACE-PE plasmid, and b represents the verification of each element when targeting the two sites FANCF and HEK3.

[0028] Figure 2 This is a schematic diagram of the verification results of the editing functions and efficiency of the PACE-PE editor, where a represents the composition of PE7max and Cas9n-D10A nickase in the PACE-PE editor, b represents the editing efficiency of the PACE-PE editor, and c represents the insertion and deletion frequency of the PACE-PE editor.

[0029] Figure 3 This is a schematic diagram of the protein language optimization results from the PACE-PE editor.

[0030] Figure 4 This is a schematic diagram showing the editing efficiency results of the PACE-PE7max and enPACEP-PE7max editors at different endogenous sites.

[0031] Figure 5 This is a schematic diagram showing the insertion and deletion frequency of the enPACE-PE editor and the editing specificity of scaffold byproducts, where a represents the insertion and deletion frequency of the editor and b represents the scaffold insertion frequency of the editor. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Example 1: Construction of the PACE-PE System

[0036] 1. Construction of PACE-PE plasmid

[0037] First, a lead editor, PE7max, based on Cas9n-H840A (SEQ ID NO: 1), MMLV reverse transcriptase (SEQ ID NO: 2), and LA protein (SEQ ID NO: 3), was constructed. Then, a nicking enzyme based on Cas9n-D10A (SEQ ID NO: 4) was constructed. The PACE-PE editor is constructed using PE7max in conjunction with the Cas9n-D10A nicking enzyme (e.g., ...). Figure 1As shown in Figure a), we first synthesized the gene fragments of the key components, designed the primers for construction, and used the RNF2sgRNA expression plasmid as a template for all gRNA expression plasmids.

[0038] (1) gRNA construction: Using RNF2 sgRNA expression plasmid as a template, enzyme digestion and ligation were performed using a system of 0.25 μL EC3IL enzyme, 0.25 μL T4 ligase, 0.5 μL T4 ligase buffer, and 0.1 μL BSA. The reaction conditions were 37℃ for 3 min, 25℃ for 4 min, 35 cycles, 80℃ for 5 min, and 25℃ for 5 min. Transformation was performed using Trans-T1 competent cells. The transformation process was: ice bath for 10 min, 42℃, water bath for 2 min, ice bath for 5 min, 500 μL LB solution was added, and amplification was performed on a shaker at 220 r / min and 37℃ for 30 min. Then, centrifugation was performed at 12000 r / min for 2 min, and the amplified cells were plated on LB solid plates with AMP resistance. Single colonies were picked and sent for testing the next day.

[0039] (2) Construction of pegRNA (pegRNA consists of primer binding site (PBS), RT template (RTT), spacer sequence, scaffold sequence and plasmid backbone): Based on the gRNA construction, PCR was performed using Prime Star. The system was 50 μL, consisting of 25 μL Prime Star, 1 μL F / R primer, 2 μL DMSO, 80 ng template (RNF2 sgRNA expression plasmid was used as template), and dd water to make up the volume. The reaction conditions were 98℃ for 3 min, 98℃ for 15 s, 62℃ for 15 s, 72℃ for 4 min, and 35 cycles were set according to the fragment length, followed by 72℃ for 5 min and 25℃ for 5 min. Ligation was performed using homologous recombinase, with the reaction conditions being 50℃ for 1 h and 4℃ for 10 min. Transformation was performed using Trans-T1 competent cells. The transformation process was as follows: 10 min on ice, 2 min in a 42°C water bath, 5 min on ice, 500 μL of LB solution was added, and the cells were amplified for 30 min on a shaker at 220 rpm and 37°C. The cells were then centrifuged at 12000 rpm for 2 min and plated onto LB agar plates containing AMP resistance. Single colonies were picked and tested the following day.

[0040] (3) Editor construction: PCR was performed using Prime Star in a 50 μL system, consisting of 25 μL Prime Star, 1 μL F / R primer, 2 μL DMSO, 80 ng template, and dd water to make up the volume. The reaction conditions were 98℃ for 3 min, 98℃ for 15 s, 62℃ for 15 s, and 72℃ for 35 cycles, with the time set according to the fragment length. The reaction was repeated at 72℃ for 5 min and 25℃ for 5 min. Ligation was performed using homologous recombinase under the following conditions: 50℃ for 1 h and 4℃ for 10 min. Transformation was performed using Trans-T1 competent cells. The transformation process was: ice bath for 10 min, 42℃, water bath for 2 min, ice bath for 5 min, 500 μL of LB solution was added, and amplification was performed on a shaker at 220 r / min and 37℃ for 30 min. Then, the cells were centrifuged at 12000 r / min for 2 min and plated onto LB solid plates with AMP resistance. Single colonies were picked and tested the next day to obtain the corresponding plasmids. Figure 1 Figure a shows a schematic diagram of the composition and working principle of the PACE-PE plasmid.

[0041] 2. Verification of key components of PACE-PE

[0042] First, HEK293T cells were seeded in 24-well plates at a density of 80,000 cells per well. After 24 hours, when the density reached 60%-80%, transfection was performed. The transfection process involved mixing PE7max and pegRNA, then adding CAS9-D10A or Cas9n-H840A cleavage enzymes (PE7max:pegRNA:key element = 600ng:300ng:300ng, key element being any one of Cas9n-D10A, Cas9n-H840A, dCas9, MMLV-RT, or WT-Cas9) plasmids. 50 μL of Opti and PEI (DNA to PEI mass ratio: 1:3-5) were added, and after mixing for 10 minutes, the mixture was added to the 24-well plates. The medium was changed after 6 hours. Cell pellets were collected 72 hours after transfection, and the cell genome was recovered using a genomic DNA recovery kit. PCR was performed using 2×Taq Master Mix enzymes, with one cycle at 95℃ for 3 minutes, followed by 10 seconds at 95℃ and 56℃. 10s, 72℃, 10s, 35 cycles. Library construction was then performed, and the samples were sent to a sequencing company for sequencing and analyzed using a CRISPResso2 sequencing system. Results are as follows: Figure 1 The results in b show that we targeted two sites, FANCF and HEK3. Among multiple combinations, only Cas9n-D10A consistently and significantly improved the PE7max-mediated editing efficiency without puromycin selection.

[0043] Example 2: Verification of PACE-PE effect

[0044] To further verify the editing functions and efficiency of the PACE-PE editor, HEK293T cells were first seeded in 24-well plates at a density of 80,000. After 24 hours, when the density reached 60%-80%, transfection was performed. The transfection process involved adding PE7max, Cas9n-D10A cleavage enzyme, and pegRNA (editor: Cas9n-D10A:pegRNA = 600ng:200ng:300ng) to 50 μL of Opti and PEI (DNA to PEI mass ratio: 1:3~5). The composition of PE7max and Cas9n-D10A cleavage enzyme is shown in the diagram below. Figure 2 As shown in Figure a, after mixing for 10 min, the mixture was added to a 24-well plate. The medium was changed after 6 h, and the cell pellet was collected 72 h after transfection. Cell genome was recovered using a genomic DNA recovery kit. PCR was performed using 2×Taq Master Mix enzyme: 95℃ for 3 min (1 cycle), followed by 35 cycles of 95℃ for 10 s, 56℃ for 10 s, and 72℃ for 10 s. Library construction was then performed, and the samples were sent to a sequencing company for sequencing. Efficiency analysis was performed using CRISPResso2. Figure 2 The results in section b show that PACE-PE improved editing efficiency at multiple sites from 1.9-22.4% when using PE7max to 14.6-68.5%, and its insertion and deletion efficiency was as follows: Figure 2 As shown in Figure c, there was no significant increase in the formation of insertions and deletions.

[0045] Example 3: Protein Language Optimization of PACEP-PE

[0046] To further optimize the functionality of PACE-PE, we used Saprot to saturate the mutations in active Cas9 and its HNH and RuvC-I / II / III domains using the SaProt_650M model. The structures of these proteins were predicted using AlphaFold3 and uploaded to SaprotHub, yielding mutation sites and scores for all proteins. Further screening was performed, selecting the top 0.5% of mutations in Cas9 and the top 5% in each domain to identify overlapping candidate mutations. These candidate mutations were then further screened using position-specific scoring matrices (PSSMs) to prioritize replacements with favorable biochemical properties. Additionally, non-overlapping mutations in the top 0.5% of Cas9 mutations were screened using PSSMs to retain high-confidence variants. We used Prime Star for PCR in a 50 μL system: 25 μL Prime Star, 1 μL F / R primer, 2 μL DMSO, 80 ng template, and dd water to make up the volume. The reaction conditions were 98℃ for 3 min, 98℃ for 15 s, 62℃ for 15 s, and 72℃ for 35 cycles (72℃ for 5 min, 25℃ for 5 min), adjusted according to fragment length. Ligation was performed using homologous recombinase at 50℃ for 1 h and 4℃ for 10 min. Transformation was performed using Trans-T1 competent cells. The transformation process involved an ice bath for 10 min, a 42℃ water bath for 2 min, an ice bath for 5 min, the addition of 500 μL of LB solution, and amplification at 220 rpm and 37℃ for 30 min on a shaker. The amplification was then performed by centrifugation at 12000 rpm for 2 min and plated onto LB agar plates containing AMP resistance. The next day, a single colony was selected for testing, thus obtaining the mutant plasmid. Subsequently, we first seeded HEK293T cells in 24-well plates at a density of 80,000. After 24 hours, when the density reached 60%-80%, we transfected the cells. The transfection process involved adding PE7max, Cas9n-D10A nickase, and pegRNA (editor: Cas9n-D10A:pegRNA = 600ng:200ng:300ng) to 50 μL of Opti and PEI (DNA to PEI mass ratio: 1:3~5), mixing for 10 minutes, and then adding the mixture to 24-well plates. After 6 hours, the medium was changed. After 72 hours of transfection, the cell pellet was collected, and the cell genome was recovered using a genomic DNA recovery kit. PCR was performed using 2×Taq Master Mix enzymes, with 1 cycle at 95℃ for 3 minutes, followed by 35 cycles at 95℃ for 10 seconds, 56℃ for 10 seconds, and 72℃ for 10 seconds. Library construction was then performed, and the samples were sent to a sequencing company for sequencing. Efficiency analysis was conducted using CRISPResso2. Ultimately, six mutation sites were selected. Figure 3This demonstrates the editing capabilities of enPACE-PE7max (Cas9n-H840A-S55E-I122D-I170N-D947G (as shown in SEQ ID NO: 5), Cas9n-D10A-N803P-D861S (as shown in SEQ ID NO: 6)) obtained after language optimization by the PACE-PE7max editor. Figure 4 The editing efficiency of enPACE-PEmax was improved at multiple endogenous sites, while... Figure 5 Figures a and b respectively verify that the enPACE-PEmax editor maintains editing specificity for insertion loss and scaffold byproducts.

[0047] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A phase-splitting nick enzyme-mediated leader editor, characterized in that, The lead editor integrates a Cas9 cleavage enzyme 1 and Cas9 cleavage enzyme 2 platform guided by a single single guide RNA, comprising Cas9 cleavage enzyme 1 and Cas9 cleavage enzyme 2, MMLV reverse transcriptase, LA protein, and pegRNA; wherein Cas9 cleavage enzyme 1 is used to achieve non-target strand cleavage, and Cas9 cleavage enzyme 2 is used to achieve target strand cleavage, and the cuts made by both are symmetrical; Cas9 cleavage enzyme 1 is selected from any one or more of the following: (i) The nickase 1 is a Cas9n-H840A mutant, and its amino acid sequence is shown in SEQ ID NO: 1; (ii) The nickase 1 is a Cas9n-H840A-S55E-I122D-I170N-D947G mutant, and its amino acid sequence is shown in SEQ ID NO: 5; The Cas9 nickase 2 is selected from any one or more of the following: (a) The nickase 2 is a Cas9n-D10A mutant, and its amino acid sequence is shown in SEQ ID NO: 4; (b) The nicking enzyme 2 is Cas9n-D10A-N803P-D861S, and its amino acid sequence is shown in SEQ ID NO:

6.

2. The preview editor according to claim 1, characterized in that, The amino acid sequence of the MMLV reverse transcriptase is shown in SEQ ID NO:2, and the amino acid sequence of the LA protein is shown in SEQ ID NO:

3.

3. The method for constructing a pilot editor as described in claim 1 or 2, characterized in that, Includes the following steps: 1) Construct editors PE7max and Cas9 nickase 2, respectively, containing Cas9 nickase 1; 2) gRNA was constructed by enzyme digestion and ligation, and then pegRNA was constructed by PCR and homologous recombination; 3) Mix PE7max with pegRNA and add Cas9 cleavage enzyme 2 to obtain PACE-PE editor.

4. The method according to claim 3, characterized in that, The enzyme digestion and ligation system for constructing gRNA includes EC3IL enzyme, T4 ligase, T4 ligase buffer and BSA, and the mass ratio of PE7max:pegRNA:Cas9 cleavage enzyme 2 is (2-6):(1-2):(1-1.5).

5. A phase-splitting nick enzyme-mediated leader editing system, characterized in that, Includes the pilot editor as described in either claim 1 or 2.

6. A method for gene editing in eukaryotic cells, characterized in that, The method described is for non-disease diagnosis and treatment purposes. Cells are transfected with the pilot editor described in claim 1 or 2, or the pilot editing system described in claim 5. Opti medium and PEI are added during the transfection process to make the DNA:PEI mass ratio 1:2~10. After transfection, the cell pellet is collected to obtain gene-edited cells.

7. The method according to claim 6, characterized in that, The DNA:PEI mass ratio is 1:3~5; the eukaryotic cells are HEK293T cells; Before transfection, the samples were cultured in 24-well plates at a rate of 6 × 10⁻⁶. 4 -10×10 4 The cells are plated at a density of 60%-80% and cultured until the cell density reaches 60%-80%. Transfection is then performed, and the medium needs to be changed 6 hours after transfection. The cell pellet is collected 70-80 hours after transfection.

8. The application of the lead editor of claim 1 or 2 or the lead editing system of claim 5 in improving the efficiency of genome lead editing.

9. The application of the lead editor of claim 1 or 2 or the lead editing system of claim 5 in reducing the proportion of insertion and deletion byproducts during genome lead editing.

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