A cytosine base editing system for improving efficiency and expanding the editing window

Abstract

The application discloses a cytosine base editing system capable of increasing a genome mutation rate and expanding an editing window, comprising a mouse-derived APOBEC1 (rA1), dDDA (E1347A), nCas9 (D10A) and a UGI sequence. A new base editing tool (BE4max-M-dDDA) is provided, which has the functions of increasing the genome mutation rate and expanding the editing window, can improve the editing efficiency and expand the editing window, and is helpful for people to have different choices when corresponding to different mutation sites and different mutation positions.

Description

Technical Field

[0001] This invention discloses a cytosine base editing system capable of increasing genome mutation rate and expanding the editing window, comprising mouse-derived APOBEC1 (rA1), dDDA (E1347A), nCas9 (D10A), and UGI sequences. A cytosine base editing system with higher mutation efficiency and a larger editing window (BE4max-M-dDDA) has been developed, expanding the target range and showing promising application prospects, belonging to the field of biotechnology. Technical Background

[0002] CRISPR / Cas9 technology introduces DNA double-strand breaks (DSBs) at target sites, enabling gene editing at specific sites and revolutionizing the biomedical field by modifying or correcting harmful genes to promote the treatment of genetic diseases. Guided by sgRNA, the Cas9 protein can reach the designated region and exert nuclease activity to cut the DNA double strand. However, directly using the Cas9 system often makes precise editing difficult. Based on CRISPR technology, more precise single-base editing systems (ABE and CBE) have been developed, capable of switching four base types (CT, GA, AG, and TC).

[0003] Currently, the commonly used CBE system BE4max has an editing window of approximately 4-8 nt at the target site, within which CT conversion can be achieved, but only a small portion of C can be edited within a 20 nt sgRNA. To improve editing efficiency and expand the editing window, more deaminases with different properties have been developed, such as A3A, A3G, and AID. Alternatively, other accessory proteins such as rad51 and Sox2 proteins can be fused. Therefore, developing editors with higher mutation efficiency and larger editing windows allows for different choices for different mutation sites and locations. Summary of the Invention

[0004] The purpose of this invention is to provide a cytosine base editing system that improves efficiency and expands the editing window, and relates to a new base editing tool (BE4max-M-dDDA) that can improve editing efficiency and widen the editing window.

[0005] This invention discloses a cytosine base editing system that improves efficiency and expands the editing window, comprising rA1, dDDA, nCas9 (D10A), and UGI; wherein dDDA is inserted into the BE4max system composed of rA1-nCas9-UGI to obtain a novel CBE system BE4max-M-dDDA of rA1-dDDA-nCas9-UGI, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0006] The present invention discloses a cytosine base editing system that improves efficiency and expands the editing window, characterized in that: compared with the BE4max system, BE4max-M-dDDA has higher editing efficiency and a wider editing window (C3-C15).

[0007] The method for preparing a cytosine base editing system that improves efficiency and expands the editing window, as described in this invention, includes the following steps: The construction of BE4max-M-dDDA and the validation of its editing efficiency in N2a cells were carried out. The dDDA construct was inserted into the BE4max vector to obtain BE4max-M-dDDA. The efficiency of BE4max-M-dDDA was validated at three N2a cell sites (DMNT1, NE2R3, VEGFA). Compared with BE4max, BE4max-M-dDDA improved the editing efficiency, and efficient editing was detected at both C14 and C15 sites.

[0008] The system window of BE4max-M-dDDA was characterized. The editing activity of BE4max-M-dDDA was evaluated at 9 sites, and the editing window was characterized as C3-C15.

[0009] By designing an early termination mutation in exon 12 of the DMD gene, it was observed at the mouse embryo level that BE4max could not edit the target C at position 13, but BE4max-M-dDDA successfully edited the target C at position 13.

[0010] The positive effects of this invention are: it provides a new base editing tool (BE4max-M-dDDA) that has the function of increasing the genome mutation rate and expanding the editing window, which can improve editing efficiency and widen the editing window, and help people to make different choices for different mutation sites and different mutation positions. Attached Figure Description

[0011] Figure 1 This invention provides a schematic diagram of the BE4max-M-dDDA base editor and BE4max, as well as an image showing the editing effect on N2a cells (a) schematic diagram of BE4max-M-dDDA and BE4max vector, b) image showing the editing efficiency of BE4max-M-dDDA and BE4max N2a cells. Figure 2 This is a comparison diagram of the BE4max-M-dDDA and BE4max editing windows of the present invention; Figure 3 This diagram illustrates the premature termination of the sgRNA in exon 12 of the mouse DMD gene, along with the Sanger sequencing results of BE4max and BE4max-M-dDDA editing. Detailed Implementation

[0012] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the materials, reagents, etc. used in the following examples are commercially available.

[0013] Example 1: Construction of BE4max-M-dDDA and validation of editing efficiency in N2a cells

[0014] Vector construction of BE4max-M-dDDA The dDDA (E1347A) fragment, with the nucleotide sequence shown in SEQ ID NO.1, was synthesized by GenScript and seamlessly cloned into the BE4max backbone vector rA1 sequence to obtain the BE4max-M-dDDA vector. Subsequently, plasmid propagation and plasmid enhancement were performed to obtain a high-purity plasmid.

[0015] 1.2 Construction of sgRNA vector Three mouse endogenous gene loci were selected, and sgRNAs were designed. A cacc sequence adapter was added to the 5' end of the upstream sequence of each sgRNA, and an aaac sequence adapter was added to the 5' end of the downstream sequence.

[0016] The synthesized sgRNA oligos are shown in Table 1: Table 1. sgRNA oligos in N2a cells DMNT1 caccaacagctctgaacgagaccc aaacgggtctcgttcagagctgtt NE2R3 caccctgtgtacctggtcccggaa aaacttccgggaccaggtacacag VEGFA cacccgcttaccttggcatggtgg aaacccaccatgccaaggtaagcg After synthesis, the upstream and downstream sides were annealed using a programmed process (95°C for 5 min, then cooled to room temperature for 30 min), and the annealed product was then linked to the feedstock. BbsI The pBluescriptSKII+U6-sgRNA(F+E) empty sgRNA backbone vector was digested with enzymes, then transformed, single clones were selected and sequenced to obtain the correct sgRNA, and high-purity plasmids were obtained through large-scale extraction.

[0017] 1.3 Cell Culture and Transfection N2a cells were seeded in DMEM medium containing 1% FBS and 1% penicillin antibiotics and cultured in a 37°C cell culture incubator containing 5% CO2. Cells for transfection were seeded in 12-well plates the day before transfection. On the second day, when the cell density reached approximately 75%, the medium was changed to 10% FBS without penicillin antibiotics. Transfection was performed using HieffTrans™ Liposomal Transfection Reagent, with 0.67 μg BE4max-M-dDDA and 0.33 μg sgRNA per well. The plasmids were mixed and diluted with 100 μl of Opti-MEM as Reagent A. Simultaneously, 3 μl of Hieff transfection reagent was diluted with 100 μl of Opti-MEM as Reagent B, and the mixture was allowed to stand for 5 min. Mix reagents A and B, mix thoroughly by pipetting, let stand for 20 min, then add dropwise to the cells to be transfected in a 12-well plate, and incubate at 37°C. Change the culture medium once after 12 h; collect the cells after 48 h and then lyse them with cell lysis buffer (Novizan OneStep Mouse Genotyping Kit).

[0018] 1.4 BE4max-M-dDDA Editing Efficiency Test Using the lysis products above as templates, PCR amplification was performed on sequences near the target site. The PCR primers are shown in Table 2. Table 2. Primers for PCR identification of N2a cell sites DMNT1 ccttcgggcatagcatggtc tatatgcctcggcatcggtcc NE2R3 aactccaaacctcctcctct acacatctttggctagcatctt VEGFA cctccgaaaccatgaactttct gctcggcccagatcgta After detecting the target band by gel electrophoresis, the amplified products were sent to Shanghai Sangon Biotech Co., Ltd. for first-generation sequencing. The PCR amplification system for the target site was as follows: 12.5 μl of 2×Taq (Tiangen), 1 μl of F (10 pmol / μl), 1 μl of R (10 pmol / μl), 1 μl of template, and ddH2O to bring the total volume to 25 μl. After sequencing, the sequencing results were analyzed for efficiency using the EditR website. The results are shown below. Figure 1 As shown in b.

[0019] Example 2: Measurement of BE4max-M-dDDA editing window

[0020] 2.1 Construction of sgRNA vector Nine endogenous gene loci in HEK293T cells were selected, and sgRNAs were designed. A cacc sequence adapter was added to the 5' end of the upstream sequence of each sgRNA, and an aaac sequence adapter was added to the 5' end of the downstream sequence. The synthesized sgRNA sequences are shown in Table 3. Table 3. sgRNA oligos in HEK293T cells EGFR-1 caccgtgctgggctccggtgcgtt aaacaacgcaccggagcccagcac EGFR-2 cacccaaagcagaaactcacatcg aaaccgatgtgagtttctgctttg EMX1 caccgacatcgatgtcctccccat aaacatggggaggacatcgatgtc Tim3 caccttctacaccccagccgcccc aaacggggcggctggggtgtagaa TYRO3 caccgcagcaacgctagtgtggcc aaacggccacactagcgttgctgc VEGFA caccgaccccctccaccccgcctc aaacgaggcggggtggagggggtc TP53 caccccttcccagaaaacctacca aaactggtaggttttctgggaagg ABCD caccggagccacaggagccgctgc aaacgcagcggctcctgtggctcc ADAR cacccaaatctgtcacattgggta aaactacccaatgtgacagatttg After synthesis, the upstream and downstream sides were annealed using a programmed process (95°C for 5 min, then cooled to room temperature for 30 min), and the annealed product was then linked to the feedstock. BbsI The pBluescriptSKII+U6-sgRNA(F+E) sgRNA backbone vector was digested with enzymes, then transformed, single clones were selected and sequenced to obtain the correct sgRNA, and high-purity plasmids were obtained through large-scale extraction.

[0021] 2.2 Cell Culture and Transfection HEK293T cells were seeded in DMEM medium containing 1% FBS and 1% penicillin antibiotics and cultured in a 37°C cell culture incubator containing 5% CO2. Cells for transfection were seeded in 12-well plates the day before transfection. On the second day, when the cell density reached approximately 75%, the medium was changed to DMEM medium containing 10% FBS but without penicillin antibiotics. Transfection was performed using HieffTrans™ Liposomal Transfection Reagent, with 0.67 μg BE4max-M-dDDA and 0.33 μg sgRNA per well. The plasmids were mixed and diluted with 100 μl of Opti-MEM as Reagent A. Simultaneously, 3 μl of Hieff transfection reagent was diluted with 100 μl of Opti-MEM as Reagent B, and the mixture was allowed to stand for 5 min. Mix reagents A and B, pipette to mix well, let stand for 20 min, then add dropwise to the cells to be transfected in a 12-well plate, and incubate at 37°C. Change the culture medium once after 12 h; collect the cells after 48 h and then lyse them with cell lysis buffer (Novizan One Step Mouse Genotyping Kit).

[0022] 2.3 BE4max-M-dDDA Editing Efficiency Test Using the lysis products above as templates, PCR amplification was performed on sequences near the target site. The amplification primers are shown in Table 4. Table 4. Primers for HEK293T cell loci PCR identification EGFR-1 cccttgtctctgtgttcttgt ggaccactgattactgtggttat EGFR-2 gatgccagtaattgcctgtttc gccagttaacgtcttccttct EMX1 ccattgcttgtccctctgt cctcctgagtttctcatctgtg Tim3 ctgtctgctagagtcacattctc gcagcagtagcttcctcttta TYRO3 gttaccattgtctggtggagag cttcttcccactccaagatgag VEGFA cccagctaccacctcct ggctggagcactgtctg TP53 ctgtccccggacgatattga tgatgggatggataaaagccca ABCD ggagcccacaaagtctaccc ctgggagaagtagaggcggt ADAR agaaggcacaatccctgtgg cctgtgtttgcaccaaatgct After detecting the target band by gel electrophoresis, the amplified products were sent to Shanghai Sangon Biotech Co., Ltd. for first-generation sequencing. The PCR amplification system for the target site was as follows: 12.5 μl of 2×Taq (Tiangen), 1 μl of F (10 pmol / μl), 1 μl of R (10 pmol / μl), 1 μl of template, and ddH2O to bring the total volume to 25 μl. After sequencing, the sequencing results were analyzed for efficiency using the EditR website, and the average efficiency was plotted. The detection results are shown below. Figure 2 As shown in a.

[0023] Example 3: Verification of mouse embryo efficiency

[0024] 3.1 sgRNA Design and Synthesis To verify the efficacy of BE4max-M-dDDA, a premature termination site was designed on exon 12 of the mouse DMD gene, with the target mutation C located at position 13. The sgRNA (cagttaggaagtcaactagt) was synthesized at GenScript Biotech.

[0025] 3.2 In vitro transcription of BE4max and BE4amx-M-dDDA Use NEB to apply BE4max and BE4max-M-dDDA NotI The target band was digested and recovered, and then BE4max and BE4max-M-dDDA mRNA were obtained by in vitro transcription according to the NEB (E2060s) instructions.

[0026] 3.3 Obtaining and microinjecting mouse fertilized eggs Four-week-old female ICR mice in good condition were selected and subjected to superovulation induction using the HCG-PMSG hormone combination method. PMSG was injected intraperitoneally, followed by HCG injection 48 hours later. After HCG injection, the mice were paired with healthy male ICR mice at a 1:1 ratio. On the second day after pairing, fertilized eggs were collected from mice with vaginal plugs. The retrieved fertilized eggs were treated with hyaluronidase, followed by washing the cells with KSOM 3-4 times. The washed fertilized egg cells were then transferred to preheated M2 injection drops for later use.

[0027] Microinjection: The in vitro transcribed sgRNA, as well as the mRNAs of BE4max and BE4max-M-dDDA, were thawed on ice. BE4max was mixed with the sgRNA, and BE4max-M-dDDA was mixed with the sgRNA. After mixing and centrifugation, the mixture was aspirated into an injection needle and microinjected. After injection, the embryos were incubated at 38.5 °C for 4 days.

[0028] 3.4 Embryo efficiency assessment The developing single embryos were aspirated into the prepared NP40 embryo lysis buffer, briefly centrifuged, and then placed in a PCR instrument for lysis according to the program of 56 °C for 60 min, 95 °C for 15 min. The lysis products were then used as templates for routine PCR amplification. PCR primers are shown in Table 5. Table 5. Primers for embryo PCR identification DMD ccaactagcatattcgacctcac cacacgcacacaatcctaca After amplification, the sample was sent to Shanghai Sangon Biotech for sequencing, and the results were as follows: Figure 3 As shown.

[0029] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A cytosine base editing system that improves efficiency and expands the editing window, characterized in that: This includes rA1, dDDA, nCas9 (D10A), and UGI; In this system, dDDA is inserted into the BE4max system composed of rA1-nCas9-UGI to obtain the cytosine base editing system rA1-dDDA-nCas9-UGI, and the nucleotide sequence of the cytosine base editing system is shown in SEQ ID NO.2.

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