Guide-editing gRNA, guide-editing system constructed based thereon and application

Abstract

The application discloses a new prime editing system. The application develops a double-RNA strategy, on the basis of using one sgRNA to mediate cleavage, an auxiliary template RNA (ActRNA:t) is introduced to pair with the target strand; on this basis, a single-targeted strand editing pegRNA (tsp-pegRNA) is further developed, the sgRNA and the ActRNA:t are simplified into a double-functional single tsp-pegRNA component, and the same effect is also achieved on the editing capacity of the upstream of the breakpoint. The application develops a new strategy of extending the target strand for uPEn, successfully realizes the editing of the upstream of the cleavage site, significantly expands the editable space of uPEn, and proves the importance of the DSB repair regulation module i53 in uPEn to improve the efficiency of the upstream editing. The new prime editing system developed by the application provides wide prospects for biomedical and agricultural applications.

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology, specifically relating to a novel guided editing system and its application in gene editing. Background Technology

[0002] A recently developed guided editor (PE) is an important precision editing tool that enables various small-scale precision edits without the need for double-strand DNA breaks or exogenous DNA templates. The PE protein is composed of a Cas9 nickase (H840A nCas9, which creates a single-strand nick only on the non-target strand of DNA) fused with a reverse transcriptase domain (RT), and works in conjunction with a guide RNA (pegRNA). The pegRNA extends the traditional 5′ sgRNA module to include a 3′ functional module containing a primer binding site (PBS) and a reverse transcription template (RTT) encoding editing information. This design gives the pegRNA dual functions as a target guide (5′ module) and an editing template (3′ extension module). After recognizing a genomic target site, the PE first cleaves the non-target strand (NTS) to create a single-strand DNA nick, exposing its 3′-OH end; then, it reverse transcribes the encoding information from the 3′ extension module of the pegRNA into the NTS strand, ultimately completing the synergistic editing of the non-target and target strands through a special DNA repair mechanism within the cell. In extensive testing across various cell systems, PE demonstrated exceptional flexibility and editing purity. However, significant room for improvement remains in its editing efficiency. At the efficiency level, the less-than-ideal coordination between PE and the cellular DNA repair mechanisms that respond to its activity may be a major limiting factor.

[0003] In addition to traditional nicking enzyme-based PEs, nuclease-based PEs (PEn) based on Cas9 double-strand cleavage activity have also been gradually developed. Recent research further demonstrates that combining PEn with regulation of the double-stranded DNA break (DSB) cellular damage repair pathway can significantly improve the precision editing efficiency of PEn. For example, in our ubiquitin variant-assisted PEn system (uPEn) developed in 2023, the engineered ubiquitin variant i53 induces 5' end trimming modification of DSB, promoting precise pairing of reverse-transcribed single-stranded DNA with downstream homologous sequences of DSB, significantly improving the efficiency of previous PEn methods. Further comparative tests also show that uPEn's precision editing efficiency at multiple sites is significantly superior to several nicking enzyme-based PEs. We subsequently used uPEn to complete challenging dual-site simultaneous small fragment knock-in / knockout editing applications in sheep embryos. These advances collectively reveal the application potential of uPEn as a highly efficient subclass of PE.

[0004] It should be noted that the double-stranded DNA breakpoint generated by uPEn is located 3 bases upstream of the NGG PAM. Due to the reverse transcription activity catalyzing primer extension at the 3'-OH end exposed by the non-target strand (NTS), the editing window of the current version of uPEn is located downstream of the DNA breakpoint (the first editing site is marked "+1"). It is worth noting that our previous research found that the editing efficiency of the initial uPEn tool was negatively correlated with the breakpoint-edit distance, with low efficiency in achieving "remote editing" at sites beyond "+6". Considering the non-saturated distribution of NGG PAM in the genome (averaging once every 8 bp for both strands), a large number of potential editing sites may be located outside the highly active editing window of PE, limiting the editing space of uPEn. Therefore, the unidirectional editing characteristics of uPEn significantly affect its applicability, especially in applications requiring precise editing of fixed genomic sites (such as pathogenic mutation repair).

[0005] To this end, we developed a novel strategy for extending the target chain (TS) in uPEn, successfully achieving upstream editing of the cleavage site and demonstrating the importance of the i53 DSB repair regulation module in uPEn in improving the efficiency of this upstream editing. We named these novel uPEn tools with upstream editing capabilities uPEn versions 3.1 / 3.2. These advancements provide a universal strategy for expanding the editing space of uPEn and have significant application value. Summary of the Invention

[0006] A common limitation of existing precision gene editing systems is that editing activity is negatively correlated with the distance between the cleavage site and the target site. Despite the potential shown by the previously developed uPEn system, it also suffers from low efficiency in remote editing (the distance of the first new base from the cleavage site > 6 bp), limiting its broader application prospects.

[0007] In existing technologies, conventional uPEn editing relies on the pairing of the template RNA region in pegRNA with the non-target strand sequence. As a result, its editing range is always downstream of the DSB breakpoint, making it difficult to effectively edit sites that are far downstream of the DSB breakpoint.

[0008] This invention attempts to overcome the limitation of the original uPEn, which only directly extends the non-target strand (NTS), and explores ways to expand the editing window of uPEn by designing reverse transcription extension of the target strand (TS).

[0009] This invention aims to expand the editable space of uPEn by redesigning template RNA components to enable editing upstream of the DSB breakpoint.

[0010] Specifically, this invention first develops a dual RNA strategy: based on using a single sgRNA that mediates cleavage, an auxiliary template RNA (ActRNA:t) that partially pairs with the target strand is introduced; this template RNA, due to its partial pairing with the target strand sequence, guides direct editing upstream of the breakpoint.

[0011] Specifically, in order to simplify the above system, this invention has developed a single-strand targeted pegRNA (tsp-pegRNA) that also achieves the ability to edit upstream of the breakpoint.

[0012] This invention discloses a guided editing system, which includes:

[0013] 1) Nuclease type guide editor (PEn);

[0014] 2) sgRNA, which guides the PEn;

[0015] 3) An auxiliary template RNA molecule ActRNA:t, wherein ActRNA:t comprises a spacer sequence at the 5' end, a backbone sequence that binds to the PEn, and a 3' template sequence that is partially complementary to the target strand (TS); the guided editing system edits upstream of the double-strand break (DSB) site by mediating the reverse transcription of TS.

[0016] This invention discloses a guided editing system, which includes:

[0017] 1) Nuclease type guide editor (PEn);

[0018] 2) Target strand (TS) editing of pegRNA molecules (tsp-pegRNA), wherein the tsp-pegRNA is a fusion molecule of sgRNA and template RNA sequence, wherein the sgRNA end guides the PEn, and the 3′ end of the template RNA is partially complementary to the target strand (TS); the guided editing system edits upstream of the double-strand break (DSB) site by mediating the reverse transcription of TS.

[0019] Preferably, the length of the 5' end spacer region is 14-16 bases.

[0020] Preferably, the length of the 5' end spacer region is 15 bases.

[0021] Preferably, the 3′ end template sequence includes, in sequence, a complementary region 1, an edit sequence, and a complementary region 2, wherein the length of complementary region 1 and complementary region 2 is 12-20 bases each, and the length of the edit sequence is 1-20 bases.

[0022] Preferably, the length of the complementary region 1 is 20 bases.

[0023] Preferably, the length of the edited sequence is 1-6 bases.

[0024] Preferably, the length of the edited sequence is 2-6 bases.

[0025] Preferably, the length of the edited sequence is 1-18 bases.

[0026] Preferably, the edited sequence is a substitution of 1-6 bases in length.

[0027] Preferably, the edited sequence involves the deletion of 2-6 bases.

[0028] Preferably, the length of the edited sequence is 1-18 bases.

[0029] Preferably, the complementary region 2 has a length of 13 bases.

[0030] Preferably, the spacer region is complementary to the non-targeted strand (NTS) sequence downstream of the DSB.

[0031] Preferably, the spacer region is derived from other species and is not complementary to the edited genome sequence.

[0032] Preferably, the nuclease type guide editor is a nuclease type guide editor with Cas9 double-strand cleavage activity.

[0033] Preferably, the guided editing system further includes an i53 helper protein module.

[0034] Preferably, the sequence of the protein module is shown in SEQ ID NO.21.

[0035] This invention discloses the application of the described guided editing system in the editing of target genes in cells.

[0036] Preferably, the cells include eukaryotic cells and prokaryotic cells.

[0037] Preferably, the editing includes insertion, replacement, or deletion.

[0038] This invention first designed an ActRNA:t helper RNA component that synergizes with uPEn / sgRNA cleavage activity. Figure 2 , Figure 3 Although this RNA component is structurally similar to pegRNA, it is specifically responsible for guiding the reverse transcription and elongation of the TS strand at the DSB site mediated by uPEn / sgRNA.

[0039] This invention further simplifies sgRNA and ActRNA:t into a bifunctional single tsp-pegRNA component. Figure 4 , Figure 5 ).

[0040] The two improved schemes disclosed in this invention (sgRNA / ActRNA:t combination and tsp-pegRNA) have successfully achieved effective editing of multiple sites that are difficult to edit with ordinary uPEn / pegRNA, significantly expanding the editable space of uPEn. Figure 6 , Figure 7 , Figure 8 ); and both systems support insertion editing ( Figure 9 ), delete and edit ( Figure 10 ) and Replace Edit ( Figure 11 The editing efficiency is basically the same.

[0041] This invention names the two newly developed uPEn methods that mediate editing in the upstream direction of the breakpoint as uPEn3.1 and uPEn3.2, respectively.

[0042] This invention further reveals that the i53 module plays an important role in promoting accurate editing of uPEn3.2 and reducing editing byproducts. Figure 12 We have noted that other recent studies have attempted to combine pegRNAs with Cas12a (or Cas9)-derived pEn for TS strand editing; however, the relatively low level of accurate editing in these studies may stem from the lack of use of auxiliary modules similar to i53. This invention not only develops different guide RNA component constructions to expand the uPEn editing space, but also demonstrates that the i53 module in this novel editing system plays a crucial auxiliary role in the TS strand editing process.

[0043] The new methods developed in this study offer new possibilities for biomedical and agricultural applications.

[0044] This invention develops a novel strategy for extending the target chain (TS) in uPEn, successfully achieving upstream editing of the cleavage site, and confirms the importance of the DSB repair regulation module i53 in uPEn in improving the efficiency of this upstream editing. Attached Figure Description

[0045] Figure 1(a) Schematic diagram illustrating the low efficiency of conventional uPEn / pegRNA editing at locations far from the cleavage site (marked in red). The diagram shows the formation of DSB (DNA double-strand break) and reverse transcriptase-dependent NTS (non-target strand) extension. (b) Schematic diagram of uPEn's potential adaptation to TS strand editing. The diagram shows DSB generation, potential primer extension to the TS strand, and the completion of the final editing. Both NTS (non-target strand) and TS are labeled. The upstream position of the DSB is numbered starting from "-1". The uPEn designation represents PEN+i53, although the regulatory role of i53 is not shown in the diagram. uPEn's TS modification capability can complement the traditional NTS editing framework, thereby significantly expanding the editable region.

[0046] Figure 2 This paper presents the technical framework of the ActRNA:t helper component for enabling TS editing. A cleaving sgRNA (alternative to pegRNA) is used to guide uPEn to generate DSB. The ActRNA:t is constructed using a pegRNA-like structure containing a shortened 15-bp spacer (non-cleaving) that targets the reverse strand sequence near the sgRNA binding site to achieve a potential anchoring effect to assist subsequent operations. Two designs were employed: the left side represents the "anchored form," where the 15-nt spacer matches the NTS strand sequence near the downstream of the DSB; the right side represents the "unanchored form," where the 15-nt sequence originates from another species and has no direct match with the genomic sequence. Simultaneously, the 3' template sequence of ActRNA:t is designed based on the TS (not NTS) characteristics of the upstream uPEn / sgRNA-dependent DSB, with a 13-base complementary region 2+ editing sequence + 20-base complementary region 1 from the 3' end upstream. The synergistic effect of sgRNA and ActRNA:t guides uPEn to synthesize the edit into the TS.

[0047] Figure 3 Four genomic loci were selected, and uPEn / sgRNA / ActRNA:t-induced upstream insertions at the breakpoint -1 were designed. A 15-bp spacer in ActRNA:ts was designed to target the region downstream of the cleavage site, or a sequence derived from sheep (which does not directly match the human genome). The data presented represent three biological replicates [mean ± standard deviation].

[0048] Figure 4The diagram illustrates the design of a monomeric tsp-pegRNA. The dual RNA combination of sgRNA and ActRNA:t is simplified into a non-classical pegRNA (i.e., tsp-pegRNA). The target site of the tsp-pegRNA is determined by the 5' spacer module, while its 3' template region is designed based on the TS characteristics at the DSB. Starting from the 3' end upstream, it consists of a 13-base complementary region 2 + an edit sequence + a 20-base complementary region 1, mediating the synthesis of the edit into the TS strand.

[0049] Figure 5 .tsp-pegRNAs were designed to directly insert sequences at specific locations by guiding TS strand extension. The 3′ template sequences of these tsp-pegRNAs, from the 3′ end upstream, consist of a 13-base complementary region 2 + an editing sequence + a 20-base complementary region 1. The editing performance of each uPEn / tsp-pegRNA combination was tested in HEK293T cells (n = 3 biological replicates, mean ± standard deviation).

[0050] Figure 6 The diagram illustrates the relative distances between the target site and its upstream and downstream cleavable sites (cleavable sites are indicated by dashed lines), meaning the target site is closer to its downstream cleavable site and farther from its upstream cleavable site. Therefore, NTS chain editing and TS chain editing correspond to distal (light red solid circle) and proximal (light blue) editing, respectively.

[0051] Figure 7 Three target sites were selected. The sites were characterized by being close to their downstream potential cleavage sites (~2 bp) and far from their upstream potential cleavage sites (~9 bp). Editing was performed using either standard uPEn / pegRNA or uPEn / sgRNA / ActRNA:t. Results are presented as a bar chart on the right (n = 3 biological replicates, mean ± standard deviation). A two-tailed t-test was used to compare the difference in accurate editing levels between the sgRNA / ActRNA:t group and the pegRNA group (***P < 0.001, ****P < 0.0001).

[0052] Figure 8 Two target sites (SHANK3 and RUNX1) were selected, both located close to their downstream cleavable sites and far from their upstream cleavable sites. pegRNA and tsp-pegRNA were used to guide distal and proximal uPEn editing, respectively. Editing efficiency was determined in HEK293T cells (n = 3 biological replicates, mean ± standard deviation). The difference in accurate editing levels between the pegRNA and tsp-pegRNA groups was analyzed using a two-tailed t-test (***P < 0.001, ****: P < 0.0001).

[0053] Figure 9The insertion editing efficiency of sgRNA / ActRNA:t and tsp-pegRNA-adapted uPEn (named uPEn3.1 and uPEn3.2, respectively) at four sites was compared. Experiments were conducted in HEK293T cells (n = 3 biological replicates [except for the DNMT1-uPEn3.1 group, which had 2 replicates], mean ± standard deviation).

[0054] Figure 10 The deletion and editing efficiencies of sgRNA / ActRNA:t and tsp-pegRNA-adapted uPEn (named uPEn3.1 and uPEn3.2, respectively) at four sites were compared. Experiments were conducted in HEK293T cells (n = 3 biological replicates, mean ± standard deviation).

[0055] Figure 11 The substitution editing efficiency of uPEn adapted to sgRNA / ActRNA:t and tsp-pegRNA (named uPEn3.1 and uPEn3.2, respectively) at four sites was compared. Experiments were conducted in HEK293T cells (n = 3 biological replicates, mean ± standard deviation).

[0056] Figure 12 Comparison of the proximal editing efficacy of .tsp-pegRNA in combination with uPEn (containing i53) or PEN (without i53) at four genomic loci (HEK293T cells, n = 3 biological replicates, mean ± standard deviation). Two-tailed Student's t-test was used to determine the difference in accurate editing levels between the uPEn and PEN groups (**: P < 0.001, ***: P < 0.001, ****: P < 0.0001).

[0057] Figure 13 The effects of .tsp-pegRNA combined with uPEn, or simultaneously with the DNA non-precise repair pathway inhibitor AZD7648, on insertion or deletion editing were compared at four genomic loci (GFAP or VEGFA, insertion of 3 or 5 bp; MECP2 or CCR5, deletion of 3 or 5 bp). Experiments were conducted in HEK293T cells (n = 3 biological replicates, mean ± standard deviation). Detailed Implementation

[0058] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0060] The pGL3-U6-sgRNA-EGFP vector in the following examples was obtained from Addgene, catalog number 107721; the pGL3-U6-sgRNA-BFP vector was obtained from Addgene, catalog number 107722. The protein sequence of the i53 protein module is MLIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLAFAGKSLEDGRTLSDYNILKDSKLHPLLRLR (SEQ ID NO. 21). The PEn vector is expressed driven by the pCMV promoter and includes, in sequence, the pCMV promoter sequence, the nCas9 coding gene sequence, and the M-MLV coding gene sequence. The uPEn vector is expressed driven by the pCMV promoter and includes, in sequence, the pCMV promoter sequence, the nCas9 coding gene sequence, the M-MLV coding gene sequence, and the 53BP1 repressive ubiquitin variant sequence. The PEn and uPEn vectors were reported in (Li et al. Nature communications (2023) 14(1):305, Development of a versatile nuclease prime editor with upgraded precision).

[0061] I. Carrier Construction

[0062] To construct various guide RNA plasmids (including pegRNA, sgRNA, ActRNA:t, and tsp-pegRNA), we employed different PCR strategies based on DNA fragment size: Phanta FlashMaster Mix (Vazyme) was used for amplification products exceeding 1 kb; Phanta Max Master Mix (Vazyme) was used for fragments smaller than 1 kb. The backbones of all guide RNA plasmids were obtained by amplifying the pGL3-U6-sgRNA-EGFP plasmid (Addgene#107721). The assembly process for various pegRNA or sgRNA expression cassettes was as follows: first, primers were customized, and after annealing, extension was performed to obtain guide RNA cassette components. Subsequently, the expression cassettes were integrated into the backbone vector using MultiF Seamless Assembly Mix (ABclonal). The total volume of the assembly reaction system was set at 20 μL, including 10 μL of enzyme mixture, and the amounts of long and short fragments were calculated at 0.02 × bases and 0.04 × bases, respectively (unit: nanograms).

[0063] II. Cell Culture and Manipulation

[0064] HEK293T cells were cultured in DMEM medium (Gibco) supplemented with 10% (v / v) fetal bovine serum (FBS, Gibco). Cells were cultured routinely at 37°C in a 5% CO2 incubator. When cell confluence reached approximately 80%, cells were passaged (every 2-3 days). The transfection procedure was as follows: cells were seeded in 24-well plates. After approximately 16-24 hours of adhesion (density approximately 60%), HEK293T cells were transfected using EZ Trans transfection reagent (Life-iLab). In the sgRNA and ActRNA:t related experiments, the transfection system contained 900 nm of uPEn plasmid, 300 nm of sgRNA plasmid, and 300 nm of ActRNA:t plasmid; while in the pegRNA and tsp-pegRNA experiments, the transfection system consisted of 900 nm of uPEn plasmid or PEN plasmid, and 300 nm of pegRNA plasmid or tsp-pegRNA plasmid.

[0065] III. Flow Cytometry Cell Sorting

[0066] Three days after transfection, cells were collected and sorted using a BD Aria III flow cytometer. Sorting was based on the EGFP marker on pegRNA, ActRNA:t, or tsp-pegRNA plasmids, and in some cases, the BFP marker on sgRNA plasmids was also used. For groups containing only the EGFP marker, EGFP+ gate sorting was used; for groups containing both EGFP and BFP markers, EGFP+BFP+ gate sorting was used. Finally, 10,000 positive cells were collected by flow cytometry sorting (FACS) for subsequent genomic DNA preparation.

[0067] IV. Genomic DNA Sample Collection

[0068] Cells were collected and transferred to 50 μL of cell lysis buffer for further processing. The lysis buffer was prepared as follows: 50 μL of 1M Tris-HCl (pH 8.0), 25 μL of 10% SDS solution, and 200 μL of 20 mg / mL proteinase K solution were added, and water was added to a final volume of 5 mL. Cell samples were lysed at 37°C for 1 hour, followed by incubation at 80°C for 30 minutes. Finally, the target gene locus was amplified by polymerase chain reaction (PCR). The products were then sent to a commercial service for next-generation sequencing (NGS) analysis.

[0069] V. Specific Procedures for Targeted NGS Testing

[0070] For the analysis of the same editing site under different experimental conditions, we used 5' primers with different barcodes to prepare amplicones. Primer sequences used for target site amplification are detailed in the supplementary data. Primer design ensured that the Cas9 cleavage site was located near the central region of the amplicon. The amplification program used fallback PCR: annealing temperature started at 65°C and gradually decreased to 57°C in 0.5°C increments for 16 cycles; followed by 20 cycles of standard amplification. The total reaction volume was 40 μL, using 2×Phanta Max Master Mix (Dye Plus) (Vazyme). PCR products with different barcodes were mixed and purified using a DNA purification and recovery kit (Vazyme) according to the manufacturer's instructions. High-throughput sequencing experiments were performed by Genewiz or Annoroad Genetics on the Illumina NovaSeq platform (PE150 mode). Sequencing data preprocessing was performed using MiSeqReporter software (Illumina) for sample splitting. Alignment analysis of amplicon sequences with the reference genome was performed using the CRISPResso2 tool. All high-throughput sequencing data analyses were performed with quality filtering: only sequencing reads with an average quality score ≥30 were retained. Point mutation editing efficiency was quantified using the CRISPResso2 standard analysis mode with the "discard_indel_reads" parameter enabled. The editing frequency was calculated as: (Number of effective reads containing the target edit / Total number of amplicon-aligned reads) × 100%. For insertion / deletion editing analysis, the CRISPResso2 HDR (Homologous Directed Repair) mode was enabled, with the parameter "e" added to specify the expected edit sequence, and the "discard_indel_read" option activated. The accurate editing efficiency was calculated as: (Number of HDR-matched reads / Total number of amplicon-aligned reads) × 100%. In all experiments, the insertion / deletion (indel) frequency was calculated as: (Number of deleted reads / Total number of reads) × 100%. The indel quantification analysis window was set to extend 30 base pairs upstream and downstream of the editing site. The percentage of defective PEs is obtained by statistically analyzing the proportion of HDR reads that do not fully match the expected edits. The calculation formula is: number of incomplete HDR reads / total number of all amplon alignment reads × 100%.

[0071] VI. Statistical Analysis

[0072] All quantitative sample tests were performed in triplicate (in very rare cases, two replicates were used, for DNMT1-ActRNA:t group). Figure 4Data analysis was performed using GraphPad Prism v.8.0.1 software. Data are presented as mean ± standard deviation (SD). Significance of differences between groups (P-value) was determined using a two-tailed Student's t-test.

[0073] VII. Design Framework for Expanding uPEn Editable Space

[0074] Previous studies have shown that uPEn is inefficient at remote editing (where the first new base is located at a position >+6) (see diagram). Figure 1 a). We consider that the limitation of this uPEn editing window stems partly from its unidirectional "write" characteristic, meaning that the reverse transcriptase can only reverse transcribe and extend the cleaved NTS (non-target strand), determining the downstream direction of editing. To improve this limitation, we propose a technical framework for designing uPEn to reverse transcribe and extend the target strand (TS), thus "unfolding" its editing window upstream. Figure 1 (b) It can be assumed that when there are cleavable sites downstream of the intended editing location, this design has the potential to transform sites that are difficult to edit using ordinary uPEn into sites that can be efficiently edited by the new method.

[0075] VIII. Strategies for uPEn / sgRNA / ActRNA:t-dependent target chain (TS) editing

[0076] Unlike classic pegRNAs, which guide reverse transcription elongation by pairing with sequences in the non-target strand (NTS), achieving target strand (TS) elongation via uPEn requires a specially conformated template RNA. To break this down, we first consider separating the required targeting and template functions into two independent molecules: a cleaving sgRNA molecule and a helper template RNA molecule. The function of the sgRNA is self-evident: to independently guide uPEn to generate DSB at the target site. Figure 2Another auxiliary template RNA designed for the TS strand is called ActRNA:t (accessory template RNA for modifying the target strand). In the design of ActRNA:t, in addition to its template function for reverse transcription elongation of the TS strand, we also considered whether it needed to be anchored near uPEn / sgRNA-induced double-strand breaks (DSBs) to better serve as a template for repairing adjacent DNA breaks. To this end, we used a pegRNA-like structure to construct ActRNA:t. First, by shortening the 5′ spacer sequence to 15 bases, ActRNA:t had the potential to anchor uPEn to a specific site without inducing cleavage. Two design schemes were used to determine whether ActRNA:t was anchored or not: the left represents the "anchored form," where the 15-nt spacer matches the NTS strand sequence downstream of the DSB; the right represents the "unanchored form," where the 15-nt sequence comes from other species and has no direct match with the genome sequence. Figure 2 Based on this, the 3' end sequence of ActRNA:t will have the ability to pair with TS at the aforementioned sgRNA-dependent DSB (complementary region 2 is generally 13 bp in length), thereby mediating the elongation of the TS strand. Figure 2 Based on this design, the subsequently extended single-stranded DNA will have an edited sequence and a complementary region 1 (typically 20 bp in length). It is important to note that this 3′ end sequence design for ActRNA:t differs significantly from the 3′ end sequence design of traditional pegRNAs. In fact, there is an antisense and symmetry relationship between them.

[0077] Therefore, we inserted predefined sequences at breakpoints (i.e., inserting upstream from position -1) for multiple genomic loci, and constructed different vectors considering both the "anchored design" and "unanchored design" of the aforementioned ActRNA:t. After obtaining the vectors, we co-transfected these ActRNA:t with uPEn / sgRNA into HEK293T cells for editing experiments, collected genomic DNA samples, amplified the target site sequences, and analyzed the editing performance using next-generation sequencing. The results were interesting: regardless of the 15-bp spacer at the 5' end, ActRNA:t with the same template region consistently resulted in a highly consistent and accurate editing rate. Figure 3 This result demonstrates for the first time that the uPEn / sgRNA / ActRNA:t combination can edit upstream of the DSB by mediating the reverse transcription of the target strand (TS). Furthermore, our results show that ActRNA:t does not require a spacer sequence to anchor it near the breakpoint to effectively serve as a template for TS strand reverse transcription elongation.

[0078] IX. Develop a simplified uPEn strategy guided by editing pegRNA (tsp-pegRNA) from a single TS target strand.

[0079] We then considered whether uPEn's TS target strand editing could be simplified by integrating the two RNA components, sgRNA and ActRNA:t, into a single non-classical pegRNA. Figure 4 In this design, the 3' end region of a single pegRNA follows the principle of mediating TS strand reverse transcription elongation. That is, the 3' sequence of this non-classical pegRNA has antisense and symmetric characteristics compared to the 3' sequence in classic pegRNAs. We name it TS strand-programming pegRNA (tsp-pegRNA).

[0080] To verify this design principle, we selected two genomic sites to construct tsp-pegRNAs for cleavage site insertion (-1 position). Complementary region 2 was set to approximately 13 bp, and in addition to the inserted edited sequence, a separate 20 bp complementary region 1 was designed. Figure 5 Tests in HEK293T cells showed that these tsp-pegRNAs achieved moderately efficient and precise insertion at two sites, accompanied by a small amount of defective PE editing and varying degrees of insertion / deletion. Figure 5 These results confirm that uPEn, in combination with a single tsp-pegRNA, can also achieve TS strand editing. Subsequent experiments used a 20bp complementary region 1 to construct tsp-pegRNA.

[0081] 10. The newly developed uPEn / sgRNA / ActRNA:t and uPEn / tsp-pegRNA strategies can both expand the editing space of uPEn. We then explored whether sgRNA / ActRNA:t or tsp-pegRNA-mediated direct modification of the TS strand could expand the editing window of uPEn. Previous experience suggests that when editing with ordinary uPEn downstream of the breakpoint, the editing efficiency is inversely proportional to the distance between the breakpoint and the editing site. Therefore, we expect that when the target site is near its downstream potential cleavage site but far from its upstream potential cleavage site, editing upstream of the breakpoint will be within a more suitable window distance. Using these newly developed tools may transform these previously difficult-to-edit sites into more easily manipulated sites. Figure 6 ).

[0082] To this end, we first selected three genomic sites (CXCR4, EMX1, and IL2RB) for small fragment insertion. These sites are located near their downstream potential cleavage sites but far from their upstream potential cleavage sites. For each target site, we constructed sgRNA / ActRNA:t combinations matching the downstream cleavage site and classic pegRNAs matching the upstream cleavage site. Although the function of ActRNA:t does not depend on its 15-bp spacer sequence at the 5′ end, the spacer sequence of ActRNA:t in this experiment was still designed based on the potential anchoring site sequence within 65-bp downstream of the sgRNA target site. Experimental results showed that, due to the less-than-ideal location of these edits far from the upstream breakpoint, the standard uPEn / pegRNA system had low accuracy in editing at all three sites (0.6%, 0.5%, and 4.5%, respectively). However, when using the sgRNA / ActRNA:t-adapted uPEn system for upstream editing, the editing efficiency was significantly higher (10.5%, 17.0%, and 43.8%, respectively). Figure 7 These results demonstrate that the uPEn system using sgRNA / ActRNA:t can significantly expand the effective editing space previously covered by uPEn / pegRNA. Since the common uPEn / pegRNA we used is the previously developed uPEn3 version, we here name the version adapted to sgRNA / ActRNA:t uPEn3.1.

[0083] We further tested whether tsp-pegRNA could also transform sites that are difficult for pegRNA to edit into easily editable sites. We selected two genomic sites: both located far from upstream cleavable sites but near downstream cleavable sites (corresponding to +9 and -1 site editing, respectively). Unlike the inefficient distal editing of ordinary uPEn / pegRNA, uPEn / tsp-pegRNA was significantly effective in editing these sites. Figure 8 Therefore, the uPEn system using tsp-pegRNA can significantly expand the effective editing space previously covered by uPEn / pegRNA. Similar to the naming of the uPEn / sgRNA / ActRNA:t system described above, we here name the uPEn version adapted to tsp-pegRNA uPEn3.2.

[0084] We then directly compared the editing efficiency of the two editing strategies (uPEn3.1 and uPEn3.2). We also tested the insertion of 6×His tag sequences (18-bp in length) at multiple sites. These sites were all far from upstream cuttable sites but close to downstream cuttable sites. Experiments conducted in HEK293T showed that both methods effectively completed editing upstream of the breakpoint. Figure 9 Meanwhile, in multiple deletion and replacement editing site tests, uPEn3.1 and uPEn3.2 showed no significant difference in accurate editing efficiency. Figure 10 , Figure 11 XI. The Important Facilitating Role of the i53 Helper Module in tsp-pegRNA Adaptation-Based uPEn Editing (uPEn3.2)

[0085] uPEn (rather than the original PEN) integrates the i53 helper module that regulates the DSB repair pathway. To investigate the importance of the i53 helper module in uPEn for TS strand editing, we compared the performance of uPEn adapted to tsp-pegRNA with that of PEN. In multi-genomic site tests, uPEn exhibited higher precision editing efficiency and a lower indel rate. Figure 12 Statistical analysis showed that the accurate edit / insertion missing ratio was more than 2.3 times higher than that of PEn (from 0.38 to 0.86). These results indicate that the i53 module plays an important role in accurate editing upstream of breakpoints mediated by uPEn3.2.

[0086] XII. Further promoting effect of the DNA non-precise repair pathway inhibitor AZD7648 on tsp-pegRNA-adapted uPEn editing (uPEn3.2).

[0087] During uPEn3.2 editing, despite the assistance of the i53 module, numerous inaccurate insertion and deletion events still occurred while achieving accurate editing. Therefore, we considered adding the DNA inaccurate damage repair pathway inhibitor AZD7648 to uPEn3.2 to test its effect on uPEn3.2 editing. In multi-gene site insertion or deletion tests (GFAP or VEGFA, 3bp or 5bp insertion; MECP2 or CCR5, 3bp or 5bp deletion), we found that AZD7648 significantly reduced inaccurate insertion and deletion events in uPEn3.2 and significantly improved the efficiency of accurate editing. Figure 13 These results suggest an inhibitor combination regimen to further enhance uPEn3.2 editing performance.

[0088] Example 1: Preparation of sgRNAs and ActRNA:ts targeting specific sites

[0089] Four genes—CCR5, MECP2, PDCD1, and UBE3A—from the HEK 293T cell genome were selected as target editing sites. Insertion editing was performed upstream of each breakpoint using sgRNA in conjunction with ActRNA:t. The 15-bp spacer in ActRNA:t was designed to either target the region near the corresponding gene or to target a sheep sequence that, according to BLAST verification, showed no homology with the human genome (“unanchored”).

[0090] The construction process for these carriers is as follows:

[0091] 1. Reverse PCR amplification of linearized vectors

[0092] Table 1: Oligonucleotide sequences for preparing linearized vectors

[0093] sg-bone-F GTTTCAGAGCTAGAAATAGCAAGTTGAA(SEQ ID NO.1) sg-bone-R TTTCGTCCTTTCCACAAGATATATAAAGCC(SEQ ID NO.2)

[0094] The above oligonucleotides were used to amplify pGL3-U6-sgRNA-EGFP and pGL3-U6-sgRNA-BFP, and then linearized them.

[0095] PCR amplification system:

[0096] Template plasmid 1μL sg-bone-F (10μM) 1μL sg-bone-R (10μM) 1μL ddH2O 7μL Overall system 20μL

[0097] PCR amplification procedure:

[0098]

[0099] 2. Construct expression vectors for sgRNAs targeting specific sites.

[0100] Table 2: Preparation of oligonucleotide sequences of sgRNAs targeting specific sites (using CCR5 as an example)

[0101]

[0102] The above oligonucleotides were used for PCR amplification. The PCR system and procedure are shown below:

[0103] PCR amplification system:

[0104] Forward (10μM) 1μL Reverse (10μM) 1μL ddH2O 8μL Overall system 20μL

[0105] PCR amplification procedure:

[0106]

[0107] Homologous recombination ligation was performed on the linearized vector PCR product of pGL3-U6-sgRNA-BFP and the PCR product of sgRNAs. The system and procedure are as follows:

[0108] Homologous recombination system:

[0109] PCR product of pGL3-U6-sgRNA-BFP 1.2μL PCR products of sgRNAs 0.4μL ddH2O 8.4μL Overall system 20μL

[0110] Incubate at 50℃ for 30 minutes.

[0111] The ligation product was transformed into DH5α competent cells, and single clones were picked the following day for sequencing. Bacterial cultures with correct sequencing results were expanded and plasmids were extracted to obtain sgRNA expression plasmids targeting the target site.

[0112] 3. Constructing an ActRNA:t expression vector targeting the target site

[0113] Table 3: Preparation of ActRNA:t oligonucleotide sequences targeting specific sites (using 4bp insertion at the CCR5 site as an example)

[0114]

[0115] Forward and Reverse primers from the first two rows of Table 3 for each target gene were used for PCR amplification. The PCR system and procedure are shown below:

[0116] PCR amplification system:

[0117] Forward (10μM) 1μL Reverse (10μM) 1μL ddH2O 8μL Overall system 20μL

[0118] PCR amplification procedure:

[0119]

[0120] Subsequently, using the PCR products as templates, PCR amplification was performed using the Forward and Reverse primers listed in the last two rows for each target gene. The PCR system and procedure are shown below:

[0121] PCR amplification system:

[0122] Forward (10μM) 1μL Reverse (10μM) 1μL PCR product template 1uL ddH2O 7μL Overall system 20μL

[0123] PCR amplification procedure:

[0124]

[0125] After fragment amplification is complete, the linearized vector PCR product of pGL3-U6-sgRNA-GFP and the ActRNA:t PCR product are ligated by homologous recombination. The system and procedure are as follows:

[0126] Homologous recombination system:

[0127] Linearized pGL3-U6-sgRNA-GFP 1.2μL PCR product of ActRNA:t 0.4μL ddH2O 8.4μL Overall system 20μL

[0128] Incubate at 50℃ for 30 minutes.

[0129] The ligation product was transformed into DH5α competent cells, and single clones were picked the following day for sequencing. Bacterial cultures with correct sequencing results were expanded and plasmids were extracted.

[0130] Following the same method (taking the vector construction targeting the CCR5 site with a 4bp insertion as an example), TS editing systems of sgRNA and ActRNA:t targeting MECP2 (11bp insertion), PDCD1 (4bp insertion), and UBE3A (1bp insertion) were prepared respectively; the insertion positions of CCR5, MECP2, PDCD1, and UBE3A genes were -2, -3, -2, and -2 positions, respectively. The results showed that regardless of the 15-bp spacer at the 5' end, ActRNA:t with the same template region could lead to a highly consistent and accurate editing rate (see...). Figure 3 This result demonstrates for the first time that the uPEn / sgRNA / ActRNA:t combination can edit upstream of the DSB by mediating the reverse transcription of the target strand (TS). Furthermore, our results show that ActRNA:t does not require a spacer sequence to anchor it near the breakpoint to effectively serve as a template for TS strand reverse transcription elongation. We define the method of editing the TS strand using sgRNA / ActRNA:t as uPEn3.1.

[0131] Example 2: Preparation of tsp-pegRNAs targeting specific sites

[0132] Table 4: Preparation of tsp-pegRNA oligonucleotide sequences targeting specific sites (using 10bp insertion at the EGFR site as an example)

[0133]

[0134] The specific steps for constructing the tsp-pegRNA vector are the same as those for the preparation of the ActRNA:t expression vector in step one of Example 1.

[0135] We selected two genomic loci (EGFR and CTLA4) to construct tsp-pegRNAs for cleavage site insertion (-1 position). Complementary region 2 was set to approximately 13 bp, and in addition to the inserted edited sequence, a separate 20-bp complementary region 1 was designed (see...). Figure 5 Tests in HEK293T cells showed that these tsp-pegRNAs achieved moderately efficient and precise insertion at two sites, accompanied by a small amount of defective PE editing and varying degrees of insertion / deletion. Figure 5These results confirm that uPEn, in combination with a single tsp-pegRNA, can also achieve TS strand editing. Subsequent experiments used a 20-bp complementary region 1 to construct tsp-pegRNA. We define the method of editing the TS strand using tsp-pegRNA as uPEn3.2.

[0136] We then directly compared the editing efficiency of the two editing strategies (uPEn3.1 and uPEn3.2). We also tested the insertion of a 6×His tag sequence (18-bp in length) at multiple sites (FANCF, DNMT1, EMX1, and PAH). These sites were all relatively far from the upstream cleavable site but close to the downstream cleavable site. The design and vector construction of sgRNA, ActRNA:t, and tsp-pegRNA were similar to those described in the preceding sections of Examples 1 and 2. Experiments conducted in HEK293T showed that both methods effectively completed the editing of inserting an 18-bp sequence upstream of the cleavage site. Figure 9 ).

[0137] Simultaneously, we also conducted tests on uPEn3.1 and uPEn3.2 for deletion editing (CTLA4, RIT1, SHANK3, and UBE3A sites, with deletions ranging from 2bp to 5bp) and substitution editing (CTLA4, EGFR, MECP2, and UBE3A sites, with deletions ranging from 1bp to 4bp). The design and vector construction methods of sgRNA, ActRNA:t, and tsp-pegRNA were similar to those described in the preceding sections of Examples 1 and 2. There was no significant difference in the accuracy of deletion and substitution editing between uPEn3.1 and uPEn3.2. Figure 10 , Figure 11 ).

[0138] To investigate the importance of the i53 helper module in uPEn for TS strand editing, we compared the performance of uPEn (containing i53) and PEN (without i53) adapted to tsp-pegRNA. The sequence of the i53 helper protein module is shown in SEQ ID NO. 21. We targeted multiple sites (FANCF, HEXA, GFAP, and RNF2, inserting sequences from 2 bp to 12 bp, respectively) to perform TS editing. The design and vector construction of tsp-pegRNA were similar to those described in the earlier part of Example 2. In multi-genomic site tests, uPEn exhibited higher precision editing efficiency and lower indel rate ( Figure 12Statistical analysis showed that the accurate edit / insertion missing ratio was more than 2.3 times higher than that of PEn (from 0.38 to 0.86). These results indicate that the i53 module plays an important role in accurate editing upstream of breakpoints mediated by uPEn3.2.

[0139] We considered adding the DNA non-precise damage repair pathway inhibitor AZD7648 to uPEn3.2 to test whether it could further reduce non-precise insertion / deletion events. TS editing was carried out by targeting multiple sites (GFAP or VEGFA, insertion of 3bp or 5bp; MECP2 or CCR5, deletion of 3bp or 5bp). The design and vector construction of tsp-pegRNA were similar to those described in the earlier part of Example 2. In the multi-gene site insertion or deletion test, the results showed that AZD7648 could significantly reduce non-precise insertion / deletion events in uPEn3.2 and significantly improve the efficiency of accurate editing. Figure 13 These results suggest an inhibitor combination regimen to further enhance uPEn3.2 editing performance.

[0140] Example 3: Preparation of pegRNAs targeting specific sites

[0141] Traditional uPEn directly edits the non-target strand (NTS). We aim to compare the editing efficiency of traditional uPEn at a greater distance downstream of the breakpoint, and to compare it with the editing efficiency upstream of the breakpoint mediated by sgRNA / ActRNA:t or tsp-pegRNA. When designing the pegRNA, we designed a complementary region 2 of approximately 13 bp targeting the NTS sequence, and an additional 20-bp complementary region 1 in addition to the inserted editing sequence.

[0142] Table 5: Preparation of pegRNA oligonucleotide sequences targeting specific sites (using the insertion of 2 bp at position CXCR4 + 9 as an example)

[0143]

[0144] The specific steps for constructing the ordinary pegRNA vector are similar to those for preparing the ActRNA:t expression vector in step one of Example 1. When designing the pegRNA, we designed a complementary region 2 of approximately 13 bp for the NTS sequence, and designed an additional 20-bp complementary region 1 in addition to the inserted edited sequence.

[0145] We first selected three genomic sites (CXCR4, EMX1, and IL2RB) for small fragment insertion. These sites were located near their downstream potential cleavage sites (-2 positions) and far from their upstream potential cleavage sites (+9 positions). For each target site, we constructed sgRNA / ActRNA:t combinations matching the downstream cleavage site and classic pegRNAs matching the upstream cleavage site. Although the function of ActRNA:t does not depend on its 15-bp spacer sequence at the 5′ end, the spacer sequence of ActRNA:t in this experiment was still designed based on the potential anchoring site sequence within 65-bp downstream of the sgRNA target site. The design and vector construction of sgRNA and ActRNA:t were similar to those described in Example 1. Experimental results showed that, due to the less-than-ideal positions of these edits far from the upstream breakpoint, the standard uPEn / pegRNA system had low accuracy in editing at all three sites (0.6%, 0.5%, and 4.5%, respectively). However, when using the sgRNA / ActRNA:t-adapted uPEn system for upstream editing, the editing efficiency was significantly higher (10.5%, 17.0%, and 43.8%, respectively). Figure 7 These results demonstrate that the uPEn system using sgRNA / ActRNA:t can significantly expand the effective editing space previously covered by uPEn / pegRNA.

[0146] We further tested whether tsp-pegRNA could also transform difficult-to-edit sites of pegRNA into easily editable sites. We selected two genomic sites (SHANK3 and RUNX1 sites): both located far from upstream cleavable sites but adjacent to downstream cleavable sites (corresponding to +9 and -1 sites, respectively, for 1bp substitution editing). The design and vector construction of tsp-pegRNA were similar to those described in Example 2. Unlike the inefficient distal editing of ordinary uPEn / pegRNA, uPEn / tsp-pegRNA was significantly effective in editing these sites. Figure 8 Therefore, the uPEn system using tsp-pegRNA can also significantly expand the effective editing space previously covered by uPEn / pegRNA.

[0147] Example 4: Cell transfection and editing efficiency analysis (corresponding to the implementation of gene editing in all cells)

[0148] 1. Cell Culture and Manipulation

[0149] (1) The frozen HEK 293T cells were revived and cultured in 10cm culture dishes. 10mL of complete culture medium (90% high glucose DMEM + 10% fetal bovine serum + working concentration of penicillin-streptomycin) was added, and the cells were cultured at 37℃ and 5% CO2. When the cell density reached 90%, the cells were seeded into 24-well plates and cultured for a longer period.

[0150] (2) When the cell density in the 24-well plate reached approximately 70%, transfection was performed using the EZ Trans transfection reagent (Life-iLab) according to the instructions. In the sgRNA and ActRNA:t correlation experiments, the transfection system contained 900 ng uPEn plasmid, 300 ng sgRNA plasmid, and 300 ng ActRNA:t plasmid; while in the pegRNA and tsp-pegRNA experiments, the transfection system consisted of 900 ng uPEn plasmid or PEN plasmid, and 300 ng pegRNA plasmid or tsp-pegRNA plasmid.

[0151] (3) Eight hours after transfection, remove the culture medium containing the transfection reagent and add 700 μL of complete culture medium;

[0152] (4) 72 h after transfection, remove the culture medium, wash each well with 200 μL PBS solution, then digest and collect the cells into a 1.5 mL centrifuge tube, and resuspend the cell pellet with 300 μL PBS solution.

[0153] (5) The resuspended cell filter was used to form a single-cell suspension, which was then added to flow cytometry tubes. Cells were collected and sorted using a BD Aria III flow cytometer. Sorting was based on the EGFP marker on pegRNA, ActRNA:t, or tsp-pegRNA plasmids, and in some cases, the BFP marker on sgRNA plasmids was also used. For groups containing only the EGFP marker, the EGFP+ gate was used for sorting; for groups containing both EGFP and BFP markers, the EGFP+BFP+ gate was used for sorting. Finally, 10,000 positive cells were collected using flow cytometry sorting (FACS) for subsequent genomic DNA preparation.

[0154] 2. Genomic DNA Sample Collection

[0155] Cells were collected and transferred to 50 μL of cell lysis buffer for further processing. The lysis buffer was prepared as follows: 50 μL of 1M Tris-HCl (pH 8.0), 25 μL of 10% SDS solution, and 200 μL of 20 mg / mL proteinase K solution were added, and the volume was adjusted to 5 mL with water. Cell samples were lysed at 37°C for 1 hour, followed by incubation at 80°C for 30 minutes. The DNA sequence containing the target site was amplified using Phanta Max Master Mix (Dye Plus). The PCR reaction system and conditions are as follows:

[0156] Primer F 1μL Primer R 1μL Cell genome 2μL <![CDATA[ddH2O]]> 11μL

[0157] PCR procedure:

[0158]

[0159] The PCR products were purified and recovered after band specificity was detected by agarose gel electrophoresis, followed by targeted deep sequencing and editing efficiency analysis.

[0160] 3. Targeted NGS Testing Procedure

[0161] High-throughput sequencing experiments were performed by Genewiz or Annoroad Genomics using the Illumina NovaSeq platform (PE150 mode). Sequencing data preprocessing used MiSeq Reporter software (Illumina) for sample splitting. Alignment analysis of amplicon sequences with the reference genome was performed using the CRISPResso2 tool. All high-throughput sequencing data analyses were configured with quality filtering: only reads with an average quality score ≥30 were retained. Point mutation editing efficiency was quantified using the standard CRISPResso2 analysis mode with the "discard_indel_reads" parameter enabled. The editing frequency was calculated as: (Number of effective reads containing the target edit / Total number of all amplicon aligned reads) × 100%. For insertion / deletion editing analysis, the CRISPResso2 HDR (Homologous Directed Repair) mode was enabled, with the additional parameter "-e" specifying the expected edit sequence, and the "discard_indel_reads" option activated. The accurate editing efficiency is calculated as: (Number of HDR-matched reads / Total number of amplicon-aligned reads) × 100%. In all experiments, the insertion / deletion (indel) frequency is calculated as: (Number of removed reads / Total number of reads) × 100%. The indel quantitative analysis window is set to extend 30 base pairs upstream and downstream of the editing site. The percentage of defective PEs is obtained by statistically analyzing the proportion of HDR reads that do not fully match the intended edit, calculated as: (Number of incomplete HDR reads / Total number of amplicon-aligned reads) × 100%.

[0162] 4. Statistical Analysis

[0163] All quantitative sample tests were performed in triplicate (in very rare cases, two replicates were used, for DNMT1-ActRNA:t group). Figure 9 Data analysis was performed using GraphPad Prism v.8.0.1 software. Data are presented as mean ± standard deviation (SD). Significance of differences between groups (P-value) was determined using a two-tailed Student's t-test.

[0164] The present invention has been illustrated through the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A guided editing system, characterized in that, The guided editing system includes: 1) Nuclease type guide editor PEn; 2) sgRNA, which guides the PEn to first complete the cleavage of the target site; 3) The helper template RNA molecule ActRNA:t, wherein ActRNA:t comprises a spacer sequence at the 5' end, a backbone sequence that binds to the PEn, and a 3' template sequence that is partially complementary to the target strand TS; the guided editing system edits upstream of the double-strand break DSB by mediating the reverse transcription of the target strand TS; The guided editing system further includes an i53 auxiliary protein module; The spacer region is complementary to the non-target strand NTS sequence downstream of the DSB, or the spacer region is from another species and is not complementary to the edited genome sequence.

2. The guided editing system according to claim 1, characterized in that, The length of the 5' end spacer region is 14-16 bases.

3. A guided editing system, characterized in that, The guided editing system includes: 1) Nuclease type guide editor PEn; 2) The target strand TS edits the pegRNA molecule tsp-pegRNA, wherein the tsp-pegRNA is a fusion of sgRNA and a 3' template sequence, wherein the sgRNA guides the PEn, and the 3' template sequence is partially complementary to the target strand TS; the guided editing system edits upstream of the double-strand break DSB by mediating the reverse transcription of the target strand TS; The guided editing system further includes an i53 helper protein module or the DNA non-precise repair pathway inhibitor AZD7648.

4. The guided editing system according to any one of claims 1-3, characterized in that, The 3' end template sequence includes, in sequence, a complementary region 1, an edit sequence, and a complementary region 2. The lengths of complementary region 1 and complementary region 2 are each 12-20 bases, and the length of the edit sequence is 1-20 bases.

5. The guided editing system according to any one of claims 1-3, characterized in that, The nuclease type guide editor is a nuclease type guide editor with Cas9 double-strand cutting activity.

6. The guided editing system according to any one of claims 1-3, characterized in that, The sequence of the i53 accessory protein module is shown in SEQ ID NO.

21.

7. The use of the guided editing system according to any one of claims 1-6 for non-therapeutic purposes in editing target genes in cells.

8. The application according to claim 7, characterized in that, The cells are selected from eukaryotic or prokaryotic cells.

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