SgRNA skeleton, prime editing system and application thereof

Abstract

The application discloses an sgRNA skeleton, a prime editing system and application thereof, and belongs to the technical field of genetic engineering. Based on sgRNA skeleton optimization of LrCas9, a high-activity sgRNA skeleton structure is developed, and a new prime editing system LrCas9-NEPE recognizing A / T-rich PAM is further developed by fusing reverse transcriptase M-MLV, so that base precision replacement can be effectively realized. Based on the prime editing system, multi-base editing and structural variation are realized in rice, and a new editing tool is provided for precision modification of A / T-rich regions.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to an sgRNA backbone, a guided editing system, and its applications. Background Technology

[0002] With the development of genome editing technology, genome editing has evolved from simple gene knockout to precise editing represented by base substitution. Base substitution at the genome level is the genetic basis of many important agronomic traits and a key target for germplasm innovation. LrCas9, a gene editing system discovered in *Lactobacillus rhamnosus*, specifically recognizes the 5'-NGAAA-3' site. LrCas9 has demonstrated applicable editing efficiency in plants. Prime editing systems are precise editing tools capable of arbitrary base changes, fragment insertions, and deletions. Currently, the main prime editing tool is SpCas9 from *Streptococcus pyogenes*, which recognizes the 5'-NGG-3' PAM site and can effectively perform prime editing of C / G-rich regions. A / T-rich regions have important biological functions, but systems capable of prime editing them are still lacking. LrCas9 can edit A / T-rich regions, but prime editing of these regions is not yet effectively achieved. Summary of the Invention

[0003] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide an sgRNA backbone, a guided editing system, and its application, using a simplified LrCas9 sgRNA backbone structure that enhances editing activity, thereby achieving precise base substitution.

[0004] The technical solution of this invention to solve the above-mentioned technical problems is as follows: An sgRNA backbone is provided, the sgRNA backbone having any of the following nucleotide sequences: (1) 115nt sgRNA backbone, the nucleotide sequence of which is shown in SEQ ID NO.1; (2) A 125nt sgRNA backbone, the nucleotide sequence of which is shown in SEQ ID NO.2; (3) 129nt sgRNA backbone, the nucleotide sequence of which is shown in SEQ ID NO.3; (4) 133nt sgRNA backbone, the nucleotide sequence of which is shown in SEQ ID NO.4; (5) 143nt sgRNA backbone, the nucleotide sequence of which is shown in SEQ ID NO.5; (6) 147nt sgRNA backbone, the nucleotide sequence of which is shown in SEQ ID NO.6.

[0005] This invention provides an application of the above-mentioned sgRNA backbone in the construction of the guided editing system LrCas9-NEPE.

[0006] The present invention provides a guided editing system LrCas9-NEPE, which consists of reverse transcriptase, Cas9 nickase and pegRNA containing the above-mentioned sgRNA backbone.

[0007] Furthermore, the reverse transcriptase is Moloney murine leukemia virus reverse transcriptase M-MLV, and its nucleotide sequence is shown in SEQ ID NO. 20; the Cas9 nickase is LrCas9 nickase, and the nucleotide sequence of LrCas9 nickase after optimization with rice codons is shown in SEQ ID NO. 21.

[0008] Furthermore, pegRNA also includes the binding site PBS responsible for binding to the target DNA and the reverse transcription template RTT sequence.

[0009] This invention provides an application of the above-mentioned guided editing system LrCas9-NEPE in editing the genome sequence of organisms or biological cells.

[0010] Furthermore, this includes editing the genome sequences of A / T-rich regions.

[0011] The present invention provides a method for generating genetically modified organisms or cells by introducing the above-mentioned guided editing system LrCas9-NEPE into the organism or cell, thereby causing base mutations, insertions or deletions in the genome sequence of the organism or cell.

[0012] This invention provides an application of the above-mentioned guided editing system LrCas9-NEPE in the preparation of products that edit the genome sequence of organisms or biological cells.

[0013] The present invention also provides a gene editing kit, comprising the above-described guided editing system LrCas9-NEPE.

[0014] This invention offers the following advantages: Based on the optimization of the LrCas9 sgRNA backbone, a highly active sgRNA backbone structure was developed. Furthermore, by fusing the reverse transcriptase M-MLV, a novel guided editing system, LrCas9-NEPE, was developed to recognize A / T-enriched PAMs, enabling precise base substitution. Based on this guided editing system, multi-base editing and structural variation creation were achieved in rice, providing a new editing tool for the precise modification of A / T-enriched regions. Attached Figure Description

[0015] Figure 1 Editing efficiency of sgRNA backbones of different lengths at endogenous gene sites in rice; Figure 2 A statistical graph showing the editing efficiency of sgRNA backbones of different lengths at the Os-AG04, Os-CG01 and Os-TG02 sites in rice endogenous genes; Figure 3 Map of the LrCas9-NEPE scaffold carrier; Figure 4 Schematic diagram of the guide editing sites for OsGRF4 and OsPK7; Figure 5 This diagram illustrates the precise deletion of the Wx gene sequence using the LrCas9-NEPE guided editing system. Figure A shows the design of the Wx gene editing site; Figure B shows the electrophoresis results of LrCas9-NEPE-mediated Wx gene deletion in rice protoplasts; Figure C shows the Sanger sequencing results of the deletion event; Figure D shows the electrophoresis results of stable T0 generation plants; and Figure E shows the Sanger sequencing results of some edited plants. Figure 6 Sanger sequencing results for mutants #02-01, #03-21, and #03-22; Figure 7 To create a new germplasm map for finely regulating amylose content in the Wx gene using the LrCas9-NEPE guided editing system; Figure A shows the starch staining results of the Wx gene-edited mutant grains; Figure B shows the expression level analysis results of the Wx gene-edited mutant; Figure C shows the amylose content determination results of the Wx gene-edited mutant; Figure D shows the total starch content determination results of the Wx gene-edited mutant. Detailed Implementation

[0016] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0017] Example 1: LrCas9 sgRNA backbone optimization method 1. Secondary structure analysis of SpCas9 and LrCas9 The inventors of this invention previously determined the LrCas9 backbone length to be between 109 nt and 153 nt through truncation experiments. Within this range, a detailed structural homology analysis was performed. By comparing the secondary structure models of SpCas9 (backbone length 92 nt) and LrCas9, it was found that the guide RNA of LrCas9 mainly consists of a dimer formed by base pairing between crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA). Analysis showed that, compared to SpCas9, the redundant sequence of LrCas9 is mainly concentrated in the pairing region between crRNA and tracrRNA, while the lengths of the first, second, and third stem loops (Stem loops 1-3) in tracrRNA are highly conserved with those of SpCas9. Therefore, the key and difficult point of simplification lies in the complementary double-stranded region of crRNA::tracrRNA, and it is necessary to maintain the integrity of the terminal stem loop structure as much as possible to ensure that it can be effectively recognized and assembled by RNA-binding proteins (such as Cas9 protein).

[0018] 2. Design of the sgRNA backbone In the process of variant design, it is not only necessary to shorten the sequence length, but also to ensure that the modified sequence can still form a stable and correct secondary structure. Specifically, it is necessary to maintain the integrity of the pairing region: ensuring that the complementary base pairing between the repeat region of crRNA and the anti-repeat region of tracrRNA forms a continuous double-stranded structure; key bulges and loop structures: during Cas9 binding, the specific bulge structure between the 5' target sequence (spacer) of crRNA and tracrRNA is crucial for stabilizing the Cas9 conformation. If the design is not appropriate, it may lead to RNA folding disorder, thereby disrupting the formation of the Cas9 / sgRNA ribonucleoprotein complex. Based on the above analysis, this invention designs a series of progressively truncated secondary structure variants. To achieve effective shortening of this region, this invention does not use simple linear truncation, but simulates the folding mode of the crRNA::tracrRNA complex. By progressively deleting bases predicted to be non-core structures or highly flexible in the crRNA::tracrRNA pairing region, and repeatedly iterating the secondary structure simulation during the design process, it is ensured that after the deletion of the sequence, the retained sequence can still refold into a conformation similar to the wild type. Six candidate skeletons with different total lengths were obtained, namely 115nt, 125nt, 129nt, 133nt, 143nt and 147nt (sequences are shown in SEQ ID NO.1-6 respectively).

[0019] 3. Functional verification of the sgRNA backbone Since subtle changes in RNA secondary structure can affect intracellular processing, Cas9 protein affinity, and stability, this is a core challenge in the optimization process. To screen for variants with both excellent structure and function, this invention constructs these backbones into backbone vectors. Three endogenous sites—Os-AG04, Os-CG01, and Os-TG02—from rice (or other indicator species) are selected as test targets, and the editing efficiency is evaluated using the aforementioned six sgRNA backbones. Through consistency analysis of editing efficiency at different targets, the optimal shortened variants that maintain structural stability while exceeding or reaching the editing activity of the original backbone (139 nt) are screened. T-DNA plasmids containing the aforementioned different backbone lengths and targets are extracted and transformed into rice protoplasts using a previously published method (Zhong, Z., Liu, G., Tang, Z. et al., (2023). Efficientplant genome engineering using a probiotic sourced CRISPR-Cas9 system. Nature Communications, 14(1), 6102.). Cells were cultured at 32℃ in the dark for 2 days, and then DNA was extracted. Primers were designed (referring to the primer design method for three sites in Zhong, Z., Liu, G., Tang, Z. et al., (2023). Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system. Nature Communications, 14(1), 6102.) to amplify the DNA sequence near the editing target site by PCR. The PCR samples were treated with restriction endonucleases overnight, and the bands were observed by electrophoresis. The bands were compared with those of the samples without restriction endonucleases. If the bands were the same size, they were considered to be the edited resistance bands. The gray values ​​were calculated using ImageJ software, and the proportion of resistance bands to all bands was statistically analyzed to determine the frequency of editing events. The electrophoresis results showed that all six sgRNA backbones could achieve effective gene editing.

[0020] Depend on Figure 1-2It was found that at the three endogenous sites Os-AG04, Os-CG01, and Os-TG02, the average editing efficiency of the 115nt backbone was 46.57%, the average editing efficiency of the 125nt backbone was 34.37%, the average editing efficiency of the 129nt backbone was 45.30%, the average editing efficiency of the 133nt backbone was 26.49%, the average editing efficiency of the 143nt backbone was 25.19%, and the average editing efficiency of the 147nt backbone was 32.41%. Among them, the editing efficiency of the 115nt and 129nt backbones was significantly improved compared with the original 139nt guide RNA backbone (average editing efficiency of 27.12%).

[0021] Example 2: Construction of the LrCas9-NEPE system skeleton carrier Existing technologies (Anzalone, AV, Randolph, PB, Davis, JR et al, (2019). Search-and-replace genome editing without double-strand breaks or donor DNA[J]. Nature, 576, 149-157.) have reported guided editing systems consisting of Moloney murine leukemia virus reverse transcriptase (M-MLV) fused with SpCas9 nickase (SpCas9-H840A) and guide editing RNA (pegRNA). The pegRNA is fused with sgRNA, a DNA binding site (PBS), and a reverse transcription template (RTT). Considering the potential differences in preferences between plants and animals, this invention first tested the effect of different positions of M-MLV on guided editing efficiency in the monocotyledonous plant rice (Zhong Z, Fan T, He Y, et al. An improved plant prime editor for efficient generation of multiple-nucleotide variations and structural variations in rice[J]. Plant Communications[2025-06-24]). Experimental results show that placing M-MLV at the N-terminus of SpCas9 results in the highest editing efficiency, and the system with the highest activity is named NEPE.

[0022] Therefore, based on the NEPE system, this invention first constructed a backbone vector pLSD520 based on the LrCas9 guide editor LrCas9-NEPE. The protein unit containing M-MLV reverse transcriptase and LrCas9 nickase (H858A) is initiated by the maize ZmUBI promoter, with nuclear localization signal sequences flanking it. Transcription of the protein coding region is terminated by the Arabidopsis HSP terminator. The pegRNA sequence is initiated by the OsU6 promoter and terminated by the TTTTTT sequence. Downstream of the OsU6 promoter is a suicide gene ccdB for cloning screening. The coding sequences of M-MLV reverse transcriptase (nucleotide sequence shown in SEQ ID NO.20) and LrCas9 nickase (H858A) were optimized using rice codons (the optimized nucleotide sequence of LrCas9 nickase is shown in SEQ ID NO.21) and synthesized by Sangon Biotech. The vector map of backbone vector pLSD520 is shown below. Figure 3 The nucleotide sequence is shown in SEQ ID NO.7; where 1-1997bp is the maize ZmUBI promoter; 2010-4232bp is the M-MLV reverse transcriptase sequence; 4233-4253bp is the nuclear localization signal; 4254-8342bp is the rice codon-optimized LrCas9 nickase (H858A); 8352-8453bp is the 3' NLS coding sequence; 8461-8710bp is the Arabidopsis HSP terminator sequence; 8720-8965bp is the OsU6 promoter sequence; and 8972-9596bp is the ccdB gene sequence.

[0023] Example 3: LrCas9-NEPE enables single / multi-base guided editing at A / T enriched PAM sites. (1) Design of gene editing target sites Download the OsGRF4 (LOC_Os02g47280), OsPK7 (LOC_Os01g027670), and Wx (LOC_Os06g04200) gene sequences from the Rice Genome Database (http: / / rice.plantbiology.msu.edu / ). Using the 5'-NGAAA-3' PAM sequence of LrCas9, design pegRNAs (using a 115nt sgRNA backbone) for OsGRF4, OsPK7, and Wx. See the schematic diagram of the guide editing site design. Figure 4 and Figure 5 A) The designed sequences were synthesized by Sangon Biotech. The specific pegRNA sequences of OsGRF4, OsPK7, and Wx are shown below: OsGRF4-pegRNA (5'→3'): ; OsPK7-pegRNA1 (5'→3'): ; OsPK7-pegRNA2 (5'→3'): ; Wx-pegRNA01 (5'→3'): ; Wx-pegRNA02 (5'→3'): ; Note: Italics represent BsaI restriction enzyme digestion and cleavage sequences; wavy lines represent sgRNA; lowercase sequences represent the sgRNA backbone; uppercase sequences represent RTT & PBS sequences; uppercase underlined sequences represent 3' motif sequences, which can prevent pegRNA degradation; TTTTTT represents the OsU6 terminator.

[0024] Specifically, OsGRF4-pegRNA achieves co-editing at five sites: C>A, G>A, G>A, T>A, and T>G, removing the miR396 binding site. OsPK7-pegRNA1 and OsPK7-pegRNA2 achieve a C>A base transversion, changing leucine at position 94 to methionine (L94M). Wx-pegRNA01 and Wx-pegRNA02 can achieve a 579 bp deletion.

[0025] (2) Construction of a guided T-DNA editing system The LrCas9-NEPE system was modified from the previously published NEPE system (Zhong Z, Fan T, He Y, et al. Animproved plant prime editor for efficient generation of multiple-nucleotide variations and structural variations in rice[J]. Plant Communications[2025-06-24]). The NEPE backbone vector pLSD456 and the synthesized LrCas9-NEPE fragment were digested with SdaI+HindIII and then ligated to obtain the backbone vector pLSD520. After being synthesized by the company, the pegRNA was assembled into the backbone vector pLSD520 to construct the final T-DNA vector, which can be used for stable transformation. The specific operation steps are as follows: The pegRNA-containing vector and the backbone vector pLSD520 were mixed in the following system: 2 μL of 10×T4 DNA Ligase Buffer (NEB), 1 μL of T4 DNA ligase (NEB), 1 μL of BsaI HF V02 (NEB), 1 μL of backbone vector pLSD520 (100 ng / μL), 1 μL of pegRNA fragment vector (100 ng / μL), and 14 μL of ultrapure water. Then, the vector was ligated and digested simultaneously for 3 h in a PCR instrument at 37℃ to complete the final vector ligation and guide the construction of the T-DNA editing system.

[0026] (3) Escherichia coli transformation Transformation: E. coli competent cells DH5α (Qingke Biotechnology) were removed from a -80℃ freezer and thawed on ice for 5 min. The entire final vector was added to 100 μL of DH5α competent cells, mixed thoroughly, and incubated on ice for 30 min. Subsequently, the cells were heat-shocked in a 42℃ metal bath for 75 s, and then incubated on ice for 2 min. The cells were then transferred to a clean bench, 1 mL of antibiotic-free LB was added, and the cells were incubated on a shaker at 37℃ for 1 h.

[0027] Spreading: Centrifuge the cultured bacterial suspension at 5000 rpm for 5 min, and resuspend the bacterial cells in 30 μL of supernatant in a clean bench (discard all other supernatant). Then spread evenly onto LB culture plates (kanamycin resistant), seal the plates, and incubate overnight in a 37°C oven.

[0028] Positive clone selection: After the culture plate has been cultured, single colonies were picked and cultured on a clean bench at 37°C in a shaker for 4 mL (LB + kanamycin resistant). Plasmid extraction was then performed using a plasmid extraction kit (Zhuangmeng Biotechnology). The extracted plasmid was sent to Sangon Biotech for sequencing confirmation using primer pc-RB (SEQ ID NO.13). Positive clones with completely correct sequencing results and the plasmids were retained for later use.

[0029] (4) Evaluation of editing efficiency of LrCas9-NEPE system and creation of mutants The editing efficiency of the LrCas9-NEPE system was evaluated using the Wx site. After transient transformation of rice protoplasts mediated by PEG-Ca2+, PCR amplification, and electrophoresis, the results showed that LrCas9-NEPE mediated highly efficient fragment deletion, with an average editing efficiency of 48.82%. Figure 5 B). The deletion event was confirmed by Sanger sequencing to be LrCas9-NEPE-mediated precise fragment deletion. Figure 5 C). Stable transformation of rice was mediated by Agrobacterium to obtain T0 generation plants. DNA was extracted for mutant detection. Amplification and sequencing were performed on the OsGRF4 gene using primers OsGRF4-F (SEQ ID NO.14) and OsGRF4-R (SEQ ID NO.15); on the OsPK7 gene using primers OsPK7-F (SEQ ID NO.16) and OsPK7-R (SEQ ID NO.17); and on the Wx gene using primers Wx-F (SEQ ID NO.18) and Wx-R (SEQ ID NO.19). One heterozygous OsGRF4 mutant and two heterozygous OsPK7 mutants (named #02-01, #03-21, and #03-22, respectively) were obtained. Figure 6 ) and 13 Wx homozygous modified mutants ( Figure 5 D-5E).

[0030] (5) Determination of total starch and amylose content The expression level of the Wx gene, total starch content, and amylose content of the obtained mutant were determined according to the existing technology (Zhong, Z., Liu, G., Tang, Z. et al., (2023). Efficientplant genome engineering using a probiotic sourced CRISPR-Cas9 system. Nature Communications, 14(1), 6102.). Figure 7As shown in A-7D, the expression levels of the Wx mutant were decreased, and the contents of amylose and total starch were also reduced. These results indicate that a new rice germplasm with finely regulated amylose content was created using the LrCas9-NEPE guided editing system.

[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An sgRNA backbone, characterized in that, The sgRNA backbone has any of the following nucleotide sequences: (1) 115nt sgRNA backbone, the nucleotide sequence of which is shown in SEQ ID NO.1; (2) A 125nt sgRNA backbone, the nucleotide sequence of which is shown in SEQ ID NO.2; (3) 129nt sgRNA backbone, the nucleotide sequence of which is shown in SEQ ID NO.3; (4) 133nt sgRNA backbone, the nucleotide sequence of which is shown in SEQ ID NO.4; (5) 143nt sgRNA backbone, the nucleotide sequence of which is shown in SEQ ID NO.5; (6) 147nt sgRNA backbone, the nucleotide sequence of which is shown in SEQ ID NO.

6.

2. The application of the sgRNA backbone of claim 1 in the construction of the guided editing system LrCas9-NEPE.

3. A guided editing system LrCas9-NEPE, characterized in that, The LrCas9-NEPE guided editing system consists of reverse transcriptase, Cas9 nickase, and pegRNA containing the sgRNA backbone of claim 1.

4. The LrCas9-NEPE guided editing system according to claim 3, characterized in that, The reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase M-MLV, and its nucleotide sequence is shown in SEQ ID NO.20; the Cas9 nickase is LrCas9 nickase, and the nucleotide sequence of the LrCas9 nickase after optimization with rice codons is shown in SEQ ID NO.

21.

5. The LrCas9-NEPE guided editing system according to claim 3, characterized in that, The pegRNA also includes a binding site PBS responsible for binding to the target DNA and a reverse transcription template RTT sequence.

6. The application of the LrCas9-NEPE guided editing system according to any one of claims 3-5 in the editing of genome sequences of organisms or biological cells.

7. The application according to claim 6, characterized in that, This includes editing the genome sequence of A / T-rich regions.

8. A method for producing genetically modified organisms or cells, characterized in that, The LrCas9-NEPE guided editing system according to any one of claims 3-5 is introduced into an organism or cell, thereby causing base mutations, insertions or deletions in the genome sequence of the organism or cell.

9. The use of the LrCas9-NEPE guided editing system according to any one of claims 3-5 in the preparation of products for editing the genome sequence of an organism or biological cell.

10. A gene editing kit, characterized in that, Includes the LrCas9-NEPE guided editing system as described in any one of claims 3-5.

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