Adenine base editing system developed by I-type CRISPR (clustered regularly interspaced short palindromic repeats) related transposase

Abstract

The invention discloses an adenine base editing system developed by utilizing I-type CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) related transposase, and develops an adenine base editing system (I-F CAST QCascade ABE) with an expanded and movable editing window, and the adenine base editing system has an expanded and movable targeting range. Compared with an ABE8e system, the target range is expanded, an editing window can be moved by changing the length of the crRNA spacer, and the application prospect is good.

Description

Technical Field

[0001] The present invention discloses an adenine base editing system developed using type I CRISPR-associated transposase, and develops an adenine base editing system (IF CAST QCascade ABE) with an expanded and movable editing window, which has an expanded and movable targeting range. Compared with the ABE8e system, the targeting range is expanded, and the editing window can be moved by changing the length of the crRNA spacer, which has good application prospects and belongs to the field of biotechnology. Technical Background

[0002] Base editors are useful for studying the effects of mutations within genes and treating genetic diseases. Current base editors mainly consist of catalytically impaired Cas9 or Cas12 orthologs fused to enzymes that change DNA bases. However, the potential for base editing using Cascades of type I CRISPR has not yet been explored. The current base editing window is limited by the size of the R-loop of Cas9 or Cas12, and Cas9 or Cas12-derived ABE8e typically has a window of less than 12 nt. Type I systems can induce larger R-loops. Such extended R-loops can provide more nucleotide substrates for ssDNA deaminases or glycosylases, may have more targetable sites, and can induce a wider variety of mutation types. A wider editing window is useful for disrupting functional sequences, such as infection-related genes or inhibitory genes for gene therapy.

[0003] The I-F3 type CRISPR-associated transposon system targets through the Cascade-TniQ complex, guiding the transposon DNA to insert into a specific target site. Unlike the typical type I CRISPR system that fixes the R-loop through the interaction between Cas11 and NTS, I-F3 fixes the R-loop through TniQ, allowing it to stably bind to DNA.

[0004] The purpose of the present invention is to explore the potential of type I systems in base editing, expand the application of type I systems in eukaryotic cells, and develop new base editors with unique editing windows. Summary of the invention

[0005] The present invention aims to provide an adenine base editing system developed using type I CRISPR-associated transposase, including the Cascade complex of Tn7016 I-F3b, TniQ transposase protein and crDNA sequence; it relates to a new base editing tool (IF CAST QCascade ABE) with a unique editing window, which expands the application of type I CRISPR system in eukaryotic cells.

[0006] The present invention discloses an adenine base editing system developed using type I CRISPR-associated transposase, including TadA-8e and IF CAST targeting elements (Cas6, Cas7, Cas8, TniQ); TadA-8e is constructed at the C-terminus of Cas6, Cas7, Cas8 and the N-terminus of TniQ, respectively. By comparison, Cas7-TadA and Cas8-TadA are co-expressed with other elements of QCascade, respectively, to produce effective editing. Among them, Cas8-TadA is co-expressed with other elements of QCascade to obtain a new ABE system IF CAST QCascade ABE with the best editing efficiency.

[0007] The present invention discloses an adenine base editing system developed using type I CRISPR-associated transposase, characterized in that: the base editing system developed based on type I CRISPR-associated transposase has a wider editing window (A6-A18) compared to the ABE8e system (A4-A8), and the editing window can be moved by changing the length of the crRNA spacer.

[0008] The preparation method of the adenine base editing system developed by the type I CRISPR-associated transposase of the present invention comprises the following steps: IF CAST QCascade ABE was constructed and the editing efficiency was verified by HEK293T cells; single expression plasmids for each element were constructed, and the TadA-8e construct was connected to the (Cas6, Cas7, Cas8, TniQ) vector to obtain Cas8-TadA, TniQ, Cas7, Cas6, TadA-TniQ, Cas7-TadA, Cas6-TadA, and Cas8 expression plasmids. The elements connected to TadA-8e were co-expressed with other QCascade elements. The first group was TadA-TniQ, Cas6, Cas7, and Cas8; the second group was TniQ, Cas7-TadA, Cas8, and Cas6; the third group was TniQ, Cas7, Cas8-TadA, and Cas6; and the fourth group was TniQ, Cas7, Cas8, and Cas6-TadA. The four combined plasmids verified the efficiency of base editing at four HEK293T cell sites (OXA1L-1, OXA1L-3, BCL11A-1, OXA1L-2). The second combination (TniQ, Cas7-TadA, Cas8, Cas6) and the third combination (TniQ, Cas7, Cas8-TadA, Cas6) could be effectively edited. Among them, the third group (TniQ, Cas7, Cas8-TadA, Cas6) achieved the best editing efficiency. The second group (TniQ, Cas7-TadA, Cas8, Cas6) was named IF CAST QCascade Cas7-TadA ABE. The third group (TniQ, Cas7, Cas8-TadA, Cas6) was named IF CAST QCascade ABE.

[0009] Characterization of the IF CAST QCascade ABE system window. The editing activity of IF CAST QCascadeABE was evaluated at 8 sites and the editing window was characterized as A6-A18.

[0010] IF CAST QCascade ABE evaluated the effect of changing the crRNA spacer length on the editing window at three sites. As the crRNA spacer length was shortened or increased, the editing window moved toward the proximal or distal end of the PAM, respectively.

[0011] The positive effect of the present invention is that it provides a new base editing tool (IF CAST QCascadeABE), a base editing system developed based on type I CRISPR-associated transposase. Compared with the ABE8e system, IF CASTQCascade ABE has a wider and adjustable editing window, which helps people have different choices when dealing with different mutation sites and different mutation positions. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the IF CAST QCascade ABE base editor of the present invention and a diagram of the editing effect of HEK293T cells (A schematic diagram of the ABE vector constructed by IF CAST QCascade, B diagram of the editing efficiency of ABE constructed by IF CAST QCascade in HEK293T cells); Figure 2 This is the IF CAST QCascade ABE editing window diagram of the present invention; Figure 3 Schematic diagram of the change in crRNA spacer length of IF CAST QCascade ABE of the present invention and a diagram of the editing effect of HEK293T cells (A schematic diagram of the change in crRNA spacer length of IF CAST QCascade ABE, B diagram of the editing efficiency of crRNA spacer length change of QCascadeABE in HEK293T). DETAILED DESCRIPTION

[0013] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0014] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0015] Example 1: Construction of IF CAST QCascade ABE and verification of editing efficiency in HEK293T cells 1.1 Construction of IF CAST QCascade ABE vector Plasmids pSL3718 (pQCascade_Pse) (#200899) and pSL3617 (pCRISPR (BbsI)_Pse) (#200902) were purchased from addgene. Single expression vectors of each component element (Cas6, Cas7, Cas8, TniQ) in the QCascade plasmid were constructed by seamless cloning. TadA-8e was then constructed at the C-terminus of Cas6, Cas7, Cas8 and the N-terminus of TniQ. PCR was performed using PrimeSTAR® Max (Takara) high-fidelity polymerase. The seamless cloning primers, fragment lengths, and PCR templates are as follows:

[0016] Afterwards, the PCR product was subjected to gel electrophoresis, the bands of corresponding sizes were cut, and the gel recovery kit was used for gel recovery; the gel recovery fragments were recombined using the Novozyme seamless cloning kit, and the recombination program was 50 ℃, 30 min. The recombinant product was then transformed using Tiangen DH5α competent cells, and the process was (ice bath for 30 min, heat shock for 90 s, ice bath for 3 min, and plate coating). Cas8-TadA, TniQ, Cas7, Cas6, TadA-TniQ, Cas7-TadA, Cas6-TadA, Cas8, and puro-Cas8-TadA expression plasmids are obtained, the nucleotide sequence of the Cas8-TadA expression plasmid is such as SEQ ID NO.1, the nucleotide sequence of the puro-Cas8-TadA expression plasmid is such as SEQ ID NO.9, the vectors of the TniQ, Cas7, Cas6, TadA-TniQ, Cas7-TadA, Cas6-TadA, and Cas8 expression plasmids are the same as SEQ ID NO.1, the expression cassette nucleotide sequences are such as SEQ ID NO.2 to SEQ ID NO.8, and the Cas8-TadA expression cassette in SEQ ID NO.1 is replaced; a high-purity plasmid is obtained by expanding the plasmid and extracting the plasmid.

[0017] 1.2 crRNA vector construction Four human endogenous gene sites were selected to design crRNA, and an AGAT sequence linker was added to the 5' end of the upstream sequence of each crRNA, and an ATTA sequence linker was added to the 5' end of the downstream sequence.

[0018] The synthesized crRNA oligos are shown in Table 1: Table 1. HEK293T cell crRNA oligos

[0019] After synthesis, the upstream and downstream were annealed by a procedure (95 °C, 5 min; room temperature cooling for 30 min), and the annealed products were connected to the upstream BbS The pSL3617 (pCRISPR (BbsI)_Pse) backbone vector was digested and then transformed, single clones were picked and sequenced to obtain the correct crRNA, and high-purity plasmids were obtained by large-scale extraction.

[0020] 1.3 Cell culture and transfection HEK293T cells were inoculated in DMEM medium containing 10% FBS. It contained 1% double antibody and was cultured in a 37 ℃ cell culture incubator containing 5% CO2. The cells used for transfection were inoculated in a 12-well plate for culture the day before. The cells were observed the next day. When the cell density was about 75%, the medium was replaced with a medium containing 10% FBS and no double antibody. HieffTransTM Liposomal Transfection Reagent was used for transfection. The total amount of each well of the 12-well plate during transfection was 1μg (the ratio of each expression vector was 1:1:1:1:1). After mixing the plasmids, dilute and mix with 100 μl Opti-MEM as reagent A. At the same time, 3 μl of Hieff transfection reagent was diluted and mixed with 100 μl Opti-MEM as reagent B, and let it stand for 5 minutes. Mix the above reagents A and B, pipette and mix well, let it stand for 20 minutes, add dropwise to the 12-well plate cells to be transfected, return to the 37°C incubator for culture, and replace with fresh culture medium after 24 hours and 48 hours. Collect the cells after 72 hours and then lyse them with cell lysis buffer (Novozyme One Step Mouse Genotyping Kit).

[0021] 1.4 IF CAST QCascade ABE editing efficiency test The above cleavage products were used as templates to perform PCR amplification on the sequences near the target site. A GGAGTGAGTACGGTGTGC sequence linker was added to the 5' end of the upstream sequence of each PCR primer, and a GAGTTGGATGCTGGATGG sequence linker was added to the 5' end of the downstream sequence for next-generation sequencing. PCR primers are shown in Table 2: Table 2. Primers for PCR identification of sites in HEK293T cells

[0022] After the target band was detected by gel electrophoresis of the amplified product, the next generation sequencing was performed. The PCR amplification system for the target site was as follows: 2×taq (Tiangen) 12.5 μl, F (10 pmol / μl) 1 μl, R (10 pmol / μl) 1 μl, template 1 μl, ddH2O filled to 25 μl. After sequencing, the efficiency analysis was performed, and the analysis results were as follows Figure 1 As shown in B.

[0023] The results showed that when the first and fourth combinations, namely TadA-8e, were connected to TniQ and Cas6, respectively, base editing was ineffective, while the second and third combinations, namely TadA-8e, were connected to Cas7 and Cas8, and effective editing was possible. Among them, the third group (TniQ, Cas7, Cas8-TadA, Cas6) achieved the best editing efficiency.

[0024] Example 2, IF CAST QCascade ABE Editing Window Determination

[0025] 2.1 Construction of crRNA vector Eight endogenous gene sites of HEK293T cells were selected to design crRNA, and an AGAT sequence linker was added to the 5' end of the upstream sequence of each crRNA, and an ATTA sequence linker was added to the 5' end of the downstream sequence. The synthesized crRNA sequences are shown in Table 3: Table 3. HEK293T cell crRNA oligos

[0026] After synthesis, the upstream and downstream were annealed by a procedure (95 °C, 5 min; room temperature cooling for 30 min), and the annealed products were connected to the upstream BbS The pSL3617 (pCRISPR (BbsI)_Pse) backbone vector was digested and then transformed, single clones were picked and sequenced to obtain the correct crRNA, and high-purity plasmids were obtained by large-scale extraction.

[0027] 2.3 Cell culture and transfection HEK293T cells were inoculated in DMEM medium containing 10% FBS. It contained 1% double antibody and was cultured in a 37 ℃ cell culture incubator containing 5% CO2. The cells used for transfection were inoculated in a 12-well plate for culture the day before. The cells were observed the next day. When the cell density was about 75%, the medium was replaced with a medium containing 10% FBS and no double antibody. HieffTransTM Liposomal Transfection Reagent was used for transfection. The total amount of each well of the 12-well plate during transfection was 1μg (the ratio of each expression vector was 1:1:1:1:1). After mixing the plasmids, dilute and mix with 100 μl Opti-MEM as reagent A. At the same time, 3 μl of Hieff transfection reagent was diluted and mixed with 100 μl Opti-MEM as reagent B, and let it stand for 5 minutes. Mix the above reagents A and B, pipette and mix well, let it stand for 20 minutes, then add dropwise to the 12-well plate cells to be transfected, return to the 37°C incubator for culture, and replace with fresh culture medium after 24 hours and 48 hours; use puro screening. Collect the cells after 72 hours and then lyse them with cell lysis buffer (Novozyme One Step Mouse Genotyping Kit).

[0028] 2.4 IF CAST QCascade ABE editing efficiency test The above cleavage products were used as templates to perform PCR amplification on the sequences near the target site. A GGAGTGAGTACGGTGTGC sequence linker was added to the 5' end of the upstream sequence of each PCR primer, and a GAGTTGGATGCTGGATGG sequence linker was added to the 5' end of the downstream sequence for next-generation sequencing. The amplification primers are shown in Table 4: Table 4. Primers for PCR identification of sites in HEK293T cells After the target band was detected by gel electrophoresis of the amplified product, the next generation sequencing was performed. The PCR amplification system for the target site was as follows: 2×taq (Tiangen) 12.5 μl, F (10 pmol / μl) 1 μl, R (10 pmol / μl) 1 μl, template 1 μl, ddH2O filled to 25 μl. After sequencing, the efficiency analysis was performed, and the analysis results were as follows Figure 2 shown.

[0029] The results show that IF CAST QCascade ABE can produce effective editing from A6 to A31, and has high editing efficiency from A6 to A18. The window of IF CAST QCascade ABE is A6-A18.

[0030] Example 3: IF CAST QCascade ABE moves the window by changing the length of crRNA

[0031] 3.1 Construction of crRNA vector Three endogenous gene sites of HEK293T cells were selected, and crRNAs with 14bp, 20bp, 26bp, 32bp, 38bp, 44bp, 50bp, 56bp, 86bp, 116bp, and 176bp spacers were designed for each site. An AGAT sequence linker was added to the 5' end of the upstream sequence of each crRNA, and an ATTA sequence linker was added to the 5' end of the downstream sequence. The synthesized crRNA sequences are shown in Table 5: Table 5. HEK293T cell crRNA oligos

[0032] After synthesis, the upstream and downstream were annealed by a procedure (95 °C, 5 min; room temperature cooling for 30 min), and the annealed products were connected to the upstream BbS pSL3617 (pCRISPR (BbsI)_Pse) backbone vector was digested. 14bp-86bp was constructed by ligation, and 116bp and 176bp were synthesized at Jiutian Gene Co., Ltd. After transformation, single clone selection and sequencing, the correct crRNA was obtained, and high-purity plasmid was obtained by large-scale extraction.

[0033] 3.2 Cell culture and transfection HEK293T cells were inoculated in DMEM medium containing 10% FBS. It contained 1% double antibody and was cultured in a 37 ℃ cell culture incubator containing 5% CO2. The cells used for transfection were inoculated in a 12-well plate for culture the day before. The cells were observed the next day. When the cell density was about 75%, the medium was replaced with a medium containing 10% FBS and no double antibody. HieffTransTM Liposomal Transfection Reagent was used for transfection. The total amount of each well of the 12-well plate during transfection was 1μg (the ratio of each expression vector was 1:1:1:1:1). After mixing the plasmids, dilute and mix with 100 μl Opti-MEM as reagent A. At the same time, 3 μl of Hieff transfection reagent was diluted and mixed with 100 μl Opti-MEM as reagent B, and let it stand for 5 minutes. Mix the above reagents A and B, pipette and mix well, let it stand for 20 minutes, then add dropwise to the 12-well plate cells to be transfected, return to the 37°C incubator for culture, and replace with fresh culture medium after 24 hours and 48 hours; use puro screening. Collect the cells after 72 hours and then lyse them with cell lysis buffer (Novozyme One Step Mouse Genotyping Kit).

[0034] 3.3 IF CAST QCascade ABE editing efficiency test The above cleavage products were used as templates to perform PCR amplification on the sequences near the target site. A GGAGTGAGTACGGTGTGC sequence linker was added to the 5' end of the upstream sequence of each PCR primer, and a GAGTTGGATGCTGGATGG sequence linker was added to the 5' end of the downstream sequence for next-generation sequencing. The amplification primers are shown in Table 6: Table 6. Primers for PCR identification of sites in HEK293T cells

[0035] After the target band was detected by gel electrophoresis of the amplified product, the next generation sequencing was performed. The PCR amplification system for the target site was as follows: 2×taq (Tiangen) 12.5 μl, F (10 pmol / μl) 1 μl, R (10 pmol / μl) 1 μl, template 1 μl, ddH2O filled to 25 μl. After sequencing, the efficiency analysis was performed, and the analysis results were as follows Figure 3 As shown in B.

[0036] The results showed that due to the particularity of type I, every 6 bases of crRNA bind to one Cas7, and shortening or lengthening the crRNA spacer by 6nt can retain effective editing. The editing window changes with the change in crRNA spacer length. As the crRNA spacer shortens, the editing window moves toward the proximal end of the PAM; as the crRNA spacer grows, the editing window extends toward the distal end of the PAM.

Claims

1. An adenine base editing system developed using type I CRISPR-associated transposase, characterized in that: The Cascade complex (Cas6, Cas8, Cas7) including Tn7016 I-F3b with TniQ transposase protein and crDNA sequence; Constructing single expression plasmids of each element, and connecting TadA-8e construction to Cas6, Cas7, Cas8, and TniQ vectors to obtain Cas8-TadA, TniQ, Cas7, Cas6, TadA-TniQ, Cas7-TadA, Cas6-TadA, and Cas8 expression plasmids, whose nucleotide sequences are shown in SEQ ID NO.1 to SEQ ID NO.8; The combination of TniQ, cas7, Cas8-TadA, and cas6 achieves the best editing efficiency and is named IF CAST QCascadeABE; compared with the ABE8e system A4-A8, IF CAST QCascade ABE has a wider editing window A6-A18, and the editing window can be moved by changing the crRNA spacer length.

2. A method for preparing an adenine base editing system developed using type I CRISPR-associated transposase as claimed in claim 1, comprising the following steps: 1) Construction of IF CAST QCascade ABE and verification of editing efficiency in HEK293T cells; Single expression plasmids of each element were constructed, and TadA-8e was constructed and connected to Cas6, Cas7, Cas8, and TniQ vectors. The elements connected to TadA-8e were co-expressed with other QCascade elements. The first group was TadA-TniQ, cas6, cas7, and cas8; the second group was TniQ, cas7-TadA, cas8 cas6; the third group was TniQ, cas7, cas8-TadA, cas6; and the fourth group was TniQ, cas7, cas8, cas6-TadA. The four combination plasmids verified the efficiency of base editing at four HEK293T cell sites OXA1L-1, OXA1L-3, BCL11A-1, and OXA1L-2. The second combination TniQ, cas7-TadA, cas8 cas6 and the third combination TniQ, cas7, cas8-TadA, cas6 could be effectively edited. Among them, the third group TniQ, cas7, cas8-TadA, and cas6 achieved the best editing efficiency; the second group TniQ, cas7-TadA, cas8 cas6 was named IF CAST QCascade Cas7-TadAABE; the third group TniQ, cas7, cas8-TadA, and cas6 was named IF CAST QCascade ABE; 2) Characterization of the IF CAST QCascade ABE system window: The editing activity of IF CAST QCascadeABE was evaluated at 8 sites and the editing window was characterized as A6-A18; 3) IF CAST QCascade ABE evaluated the effect of changing the crRNA spacer length on the editing window at three sites. As the crRNA spacer length shortened or increased, the editing window moved toward the proximal or distal end of the PAM, respectively.

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