Mitochondrial ABE base editing system capable of improving efficiency and expanding editing window

Abstract

The invention provides a mitochondrial ABE base editing system capable of improving the efficiency and expanding the editing window, provides a new base editing tool (E-sTELAD), has the functions of increasing the genome mutation rate and expanding the editing window, and can be used for exploring the function of mitochondrial DNA mutation. Meanwhile, mutated DNA can be repaired by utilizing the tool disclosed by the invention. The editing efficiency can be improved, the editing window can be widened, and people can have different choices corresponding to different mutation sites and different mutation positions.

Description

Technical Field

[0001] The present invention provides a mitochondrial ABE base editing system that improves efficiency and expands the editing window, and relates to a new base editing tool (E-sTELAD) for realizing A / T to G / C editing on the mitochondrial genome, which can improve editing efficiency and expand the editing window, and belongs to the field of biotechnology. Background Art

[0002] Mitochondrial diseases are often caused by mutations in mitochondrial DNA (mtDNA) that impair energy metabolism and other aspects of cellular homeostasis. The development of treatments for mitochondrial diseases is hampered by an incomplete understanding of the underlying biology and a lack of cell and animal models.

[0003] Therefore, tools to introduce specific modifications into mtDNA are needed to model and potentially treat these diseases. However, the development of base editing tools within mitochondrial DNA (mtDNA) has been hampered by the challenges of transporting RNA into mitochondria, including the guide RNA required for CRISPR-mediated genome editing tools.

[0004] Recently, the DddA domain of a bacterial toxin protein, DddAtox, was discovered to possess double-stranded DNA deaminase activity. By linking to TELA, it forms a cytosine base editor (DdCBE), enabling C / G to T / A mutations in mitochondria. Subsequently, it was linked to TAdA8e to form an adenine base editor (sTALAD), which can also edit A / T to G / C in mitochondria. However, the original version of sTELAD has low editing efficiency and a narrow targeting range, which significantly limits its application in disease modeling. Therefore, developing editors with higher mutation efficiency and a wider targeting window will help researchers choose the right ones for different mutation sites and locations. Summary of the Invention

[0005] The present invention aims to provide a mitochondrial ABE base editing system with improved efficiency and expanded editing window, involving a new base editing tool (E-sTELAD) for realizing A / T to G / C editing on the mitochondrial genome, which can improve editing efficiency and expand the editing window.

[0006] The present invention discloses a mitochondrial ABE base editing system with improved efficiency and expanded editing window, which is used to edit genes in animal organelles, including mitochondrial localization signal (MTS), TELA protein, Half DddA tox (G1397N, G1397C) and single-stranded DNA adenine deaminase TAdA8e, among which The MTS includes SOD2 MTS and COX8 MTS; the nucleotide sequence of the SOD2 MTS is such as SEQ ID NO.1; the nucleotide sequence of the COX8 MTS is such as SEQ ID NO.2; The TELA protein includes an N-terminal sequence, a repeating sequence that specifically recognizes the targeting sequence, and a C-terminal sequence; the nucleotide sequence of the N-terminal sequence is such as SEQ ID NO.3; the nucleotide sequence of the C-terminal sequence is such as SEQ ID NO.4; The Half DddA tox Including G1397N and G1397C; the nucleotide sequence of G1397N is such as SEQ ID NO.5; the nucleotide sequence of G1397C is such as SEQ ID NO.6; the nucleotide sequence of G1397N-Q1310R-T1380I is such as SEQ ID NO.7; the nucleotide sequence of G1397C-T1413I is such as SEQ ID NO.8; The nucleotide sequence of the single-stranded DNA adenine deaminase TAdA8e is shown in SEQ ID NO.9.

[0007] The present invention provides a mitochondrial ABE base editing system that improves efficiency and expands the editing window, characterized in that compared to the original stelad system, the E-sTELAD system has higher editing efficiency and a wider editing window (A1-A13).

[0008] The method for preparing a mitochondrial ABE base editing system with improved efficiency and expanded editing window described in the present invention comprises the following steps: According to the targeting sequence, the TELA sequences on both sides were designed according to Half DddA tox The pairing method is to select a vector that fuses the double-stranded DNA cytosine deaminase gene and the single-stranded DNA adenine deaminase TadA8e gene; the assembled vector is verified by Sanger sequencing to obtain a correctly assembled fusion vector; wherein, The Half DddA tox The pairing method is G1397N and G1397C.

[0009] The correctly assembled fusion vector was cloned according to Half DddA tox The efficiency of E-sTELAD was verified at three N2a cell sites (MT-CO1, MT-RNR1, and MT-ND1) by transfecting N2A cells in a paired manner. Compared with sTELAD, E-sTELAD improved the editing efficiency.

[0010] Characterization of the E-sTELAD system window. The editing activity of E-sTELAD was evaluated at three sites, and the editing window was characterized as A1-A13.

[0011] A D393G mutation was engineered in the MT-ND5 gene, and successful editing of the 7th target A by E-sTELAD was observed at the embryonic level.

[0012] The positive effects of this invention include providing a new base editing tool (E-sTELAD) that increases the genomic mutation rate and expands the editing window, allowing for the exploration of mitochondrial DNA mutations. Furthermore, the tool can be used to repair mutated DNA. This improves editing efficiency and widens the editing window, enabling researchers to choose the right base editing tool for different mutation sites and locations. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the working of the sTELAD and E-sTELAD systems of the present invention and the effect diagram of N2a cell editing (a) Schematic diagram of the working of the sTELAD and E-sTELAD systems, b sTELAD and E-sTELAD N2a editing efficiency diagram); Figure 2 This is a comparison diagram of the E-sTELAD and sTELAD editing windows of the present invention; Figure 3 Sequence information of the editing window designed for the D393G mutation of the mouse MT-ND5 gene of the present invention and the Sanger sequencing diagram of the E-sTELAD editing effect. DETAILED DESCRIPTION

[0014] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the techniques used in the examples are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0015] Example 1: Construction of sTELAD and E-sTELAD and Verification of Editing Efficiency in N2a Cells

[0016] Design of TELA sequences and sTELAD assembly Select 3 target ranges of mouse mitochondrial DNA and design the recognition target according to the DNA sequence near the target range. TELA sequence. Through the matching TELA assembly platform, it is assembled into the selected stelad backbone vector by seamless cloning. Then, through transformation, single clone selection and sequencing, the correct stelad vector is obtained, and high-purity plasmid is obtained through large-scale extraction.

[0017] Table 1

[0018] 1.2 E-sTELAD vector construction DddA tox Selective point mutations were made in the DddA of the stelad vector according to the selected pairing mode. tox -Q1310R and T1380I were introduced into G1397N, and DddA was introduced into the sTELAD vector. tox -T1413I was introduced into G1397C. Subsequently, the correct E-sTELAD was obtained through transformation, single clone selection and sequencing, and high-purity plasmid was obtained through large-scale extraction.

[0019] 1.3 Cell culture and transfection N2a cells were seeded in 10% FBS-containing DMEM medium containing 1% double antibody and cultured in a cell culture incubator at 37°C with 5% CO2. Cells to be transfected were plated and cultured in 12-well plates the day before. The cells were observed the next day. When the cell density reached approximately 80%, the medium was replaced with 10% FBS-free culture medium. Transfection was then performed using Hieff Trans™ Liposomal Transfection Reagent. The transfection was performed using a 1397 split combination (L-G1397N + R-G1397C-AD; R-G1397N + L-G1397C-AD) based on the DddA splitting scheme. The amount of plasmid transfection was 0.5 μg for each plasmid. The plasmids were mixed and diluted with 100 μl of Opti-MEM and mixed thoroughly as Reagent A. Simultaneously, 3 μl of Hieff transfection reagent was diluted with 100 μl of Opti-MEM and mixed thoroughly as Reagent B. The mixture was incubated at room temperature for 5 min. Reagents A and B were mixed and pipetted to mix thoroughly. After incubation at room temperature for 20 min, the mixture was added dropwise to the cells in the 12-well plate to be transfected. The cells were returned to the 37°C incubator for 12 min. Fresh culture medium was replaced after 48 h; cells were collected after 48 h to detect DNA mutation efficiency and then lysed with cell lysis buffer (Novozymes One Step Mouse Genotyping Kit).

[0020] 1.4 sTELAD and E-sTELAD editing efficiency testing The above cleavage products were used as templates to perform PCR amplification on the sequences near the target site. The PCR primers are shown in Table 2: Table 2. PCR primers for identification of N2a cell loci

[0021] After the amplified product was subjected to gel electrophoresis to detect the target band, it was sent to Shanghai Bioengineering Co., Ltd. for first-generation sequencing. The PCR amplification system for the target site was as follows: 2× Taq (Tiangen) 12.5μl, F (10 pmol / μl) 1μl, R (10 pmol / μl) 1μl, template 1μl, ddH2O filled to 25μl. After sequencing, the sequencing results were analyzed for efficiency using the EditR analysis website. The test results are as follows: Figure 1 b and Figure 2 shown.

[0022] Example 2: Mouse embryo efficiency verification

[0023] 2.1 E-sTELAD vector design and synthesis To validate the efficacy of E-sTELAD, the D393G mutation was engineered in the mouse MT-ND5 gene, with the target mutation A at position 7. The left-side recognition DNA sequence is tcctaacagggttctact; the right-side recognition DNA sequence is gcaggtattaattgcttc. The correct E-sTELAD vector was then assembled using the pairing pattern of L-G1397N-Q1310R-T1380I and R-G1397C-T1413I-AD.

[0024] 2.2 E-sTELAD in vitro transcription E-sTELAD was used by ThermoFisher Scientific NheI The target band was digested and recovered, and then E-sTELAD (MT-ND5: L-G1397N-Q1310R-T1380I and R-G1397C-T1413I-AD) mRNA was obtained by in vitro transcription according to the NEB (E2060s) instructions.

[0025] 2.3 Obtaining mouse fertilized eggs and microinjection Four-week-old, healthy female ICR mice were selected and treated with a combination of HCG and PMSG to induce superovulation. PMSG was injected intraperitoneally, followed 48 hours later by an intraperitoneal injection of HCG. Following the HCG injection, the mice were cohabitated with healthy male ICR mice at a 1:1 ratio. On the second day of cohabitation, fertilized eggs were collected from mice that had vaginal plugs. The fertilized eggs were treated with hyaluronidase and then washed three to four times with KSOM. The washed eggs were then transferred to a preheated M2 injection chamber for later use.

[0026] Microinjection: Thaw in vitro-transcribed E-sTELAD mRNA (MT-ND5: L-G1397N-Q1310R-T1380I and R-G1397C-T1413I-AD) on ice. Mix the L-G1397N-Q1310R-T1380I and R-G1397C-T1413I-AD mRNAs. Mix thoroughly, centrifuge, and draw the mixture into a needle for microinjection. After injection, incubate the embryos at 37°C for 4 days.

[0027] 2.4 Embryo efficiency assessment Aspirate a single developing embryo into the previously prepared NP40 embryo lysis buffer, centrifuge briefly, and place in a PCR instrument for lysis at 56°C for 60 minutes, then 95°C for 15 minutes. Then, use the lysis product as a template for conventional PCR amplification. PCR primers are listed in Table 3: Table 3. Embryo PCR identification primers

[0028] After the amplification, the cells were sent to Shanghai Bioengineering for sequencing. Figure 3 shown.

[0029] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may 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 one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A mitochondrial ABE base editing system with improved efficiency and expanded editing window, characterized in that: For gene editing in animal mitochondrial organelles: Including mitochondrial localization signal MTS, TELA protein, Half DddA tox , and single-stranded DNA adenine deaminase TAdA8e; among them, The MTS includes SOD2 MTS and COX8 MTS; the nucleotide sequence of the SOD2 MTS is such as SEQ ID NO.1; the nucleotide sequence of the COX8 MTS is such as SEQ ID NO.2; The TELA protein includes an N-terminal sequence, a repeating sequence that specifically recognizes the targeting sequence, and a C-terminal sequence; the nucleotide sequence of the N-terminal sequence is such as SEQ ID NO.3; the nucleotide sequence of the C-terminal sequence is such as SEQ ID NO.4; The Half DddA tox Including G1397N and G1397C; the nucleotide sequence of G1397N is as SEQ ID NO.5; the nucleotide sequence of G1397C is as SEQ ID NO.6; The nucleotide sequence of the single-stranded DNA adenine deaminase TAdA8e is shown in SEQ ID NO.

9.

2. A mitochondrial ABE base editing system for improving efficiency and expanding editing window as claimed in claim 1, characterized in that: The Half DddA tox , a single nucleotide was mutated to improve the activity of the original protein. The enhanced version of Half DddA tox The nucleotide sequence is as follows: The nucleotide sequence of G1397N-Q1310R-T1380I is shown in SEQ ID NO.7, and the nucleotide sequence of G1397C-T1413I is shown in SEQ ID NO.

8.

3. A mitochondrial ABE base editing system with improved efficiency and expanded editing window constructed according to claim 1, characterized in that: Compared with the original sTELAD system, the E-sTELAD system has higher editing efficiency and a wider editing window (A1-A13).

4. A method for preparing a mitochondrial ABE base editing system for improving efficiency and expanding editing window as described in claim 1, characterized in that The following steps are involved: 1) Design the TELA sequences on both sides according to the DNA sequence of the site to be edited, and follow the Half DddA tox The pairing method is to encode the mitochondrial localization signal peptide, the N-terminal sequence of the TELA protein, the repeat sequence of the specific recognition targeting sequence of the TELA protein and the C-terminal sequence of the TELA protein, WT-Half DddA tox or E-Half DddA with point mutation tox (G1397N-Q1310R-T1380I, G1397C-T1413I), single-stranded DNA adenine deaminase nucleic acid molecules were connected to obtain the correctly assembled vector; The efficiency of E-sTELAD was verified at three N2a cell sites (MT-CO1, MT-RNR1, and MT-ND1). Compared with sTELAD, E-sTELAD improved the editing efficiency. 2) Characterization of the E-sTELAD system window: The editing activity of E-sTELAD was evaluated at three sites and the editing window was characterized as A1-A13; 3) A D393G mutation was designed in the MT-ND5 gene, and it was observed at the mouse embryonic level that E-sTELAD successfully edited the 7th target A.

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