Cas12f nuclease with wider targeting range and derivative gene editing tool thereof

Abstract

The invention provides a Cas12f nuclease with a wider targeting range and a derivative gene editing tool of the Cas12f nuclease, successfully develops a UN1Cas12f1 mutant Cas12f-SSRSV with a wider targeting range, and evaluates the editing characteristics of the UN1Cas12f1 mutant Cas12f-SSRSV; meanwhile, an adenine base editor SSRSV-ABE, a cytosine base editor SSRSV-CBE and a transcriptional activation tool PHM-SSRSV which are wider in targeting range are constructed through an MS2-MCP strategy, and more choices are provided for target design while the problem of AAV packaging limitation is solved. The design of the invention is derived from the actual demand of a gene editing tool in clinical transformation, develops small nuclease, ABE, CBE and transcription activation tools with higher PAM compatibility, and has certain clinical transformation value.

Description

Technical Field

[0001] The present invention relates to the field of gene editing technology, and in particular to a Cas12f nuclease with a wider targeting range and a gene editing tool derived therefrom. Background Art

[0002] The CRISPR / Cas system and its derived gene editing tools have been widely used for human cell genome editing, but commonly used gene editing tools are limited by their large molecular size and are difficult to deliver efficiently into the body. The Type V-F nuclease Cas12f family is a class of nucleases found in archaea. Compared with Cas9 and Cas12a, they are very small in size, with only 400-700 amino acids. Even if they are fused with effector proteins such as deaminases and transcriptional regulators, they can be delivered through a single AAV. In addition to its small size, Cas12f also has the characteristics of high specificity, low off-target rate, and is not prone to causing large fragment deletions and chromosomal translocations. Therefore, Cas12f has broad application prospects in the field of gene therapy. However, Cas12f has low editing activity in mammalian cells, and the PAM sequence is very strict, which severely limits the application of Cas12f. At present, the research on Un1Cas12f1 has been relatively comprehensive, and its cutting mode and crystal structure have been resolved. Un1Cas12f1 recognizes 5'-TTTR-3' (R=A / G) PAM and cuts both chains almost simultaneously to form sticky ends, forming an incision 24bp downstream of the PAM of the targeted chain and a random incision 20-22bp downstream of the PAM of the non-targeted chain. Both incisions are located outside the target site. Osamu Nureki's team and Chang Leifu's team successively published articles in 2021 revealing the cryo-electron microscopy structure of Un1Cas12f1. Both articles found that Un1Cas12f1 functions as a dimer. Two Un1Cas12f1 proteins bind to an sgRNA in the form of an asymmetric homodimer to recognize and cut dsDNA.

[0003] Although Un1Cas12f1 has a size advantage, its cutting activity in mammalian cells is very low, which greatly reduces its application value. It has been previously reported that the editing efficiency of Cas9 can be improved by engineering sgRNA, so modifying sgRNA may also improve the editing efficiency of Un1Cas12f1. The Yong-Sam Kim team identified five potential modification sites (modification site, MS1-MS5) in the sgRNA of Un1Cas12f1, and finally optimized ge4.1, shortening the sgRNA from 222nt to 129nt. Compared with the original sgRNA, the length of ge4.1 was shortened by about 40%, and the editing efficiency was increased by 867 times.

[0004] In addition to optimizing sgRNA, engineering Cas proteins is also a common optimization strategy. Previous articles have reported that mutating amino acids in the DNA binding pocket can affect DNA binding and thus improve the editing efficiency of AsCas12a. Qi Lei's team rationally designed Un1Cas12f1 based on its protein structure. By comparing its structure with Cas12a, they selected 28 sites and mutated them into positively charged arginine. This strategy may increase the interaction between Un1Cas12f1 and negatively charged DNA and sgRNA. They used GFP transcriptional activation cell lines to screen 28 arginine single-point mutants, and finally optimized CasMINI V4 after multiple rounds of superposition, which significantly improved the DNA binding activity of Un1Cas12f1. Compared with wild-type Un1Cas12f1, the GFP reporter gene activation efficiency of CasMINI V4 increased by about 200 times.

[0005] Due to its small size, Un1Cas12f1 can be delivered via a single AAV even when fused with deaminases such as TadA and APOBEC1, or transcriptional regulatory elements such as VP64. Therefore, Un1Cas12f1 has great potential for application in gene therapy. Qi Lei's team fused dCasMINI V4 with the transcriptional activation element VPR to develop the transcriptional activation tool dCasMINI-VPR. They also fused adenine deaminase TadA-TadA8e to the N-terminus of dCasMINI V4 to develop dCasMINI-ABE. The editing window of dCasMINI-ABE is mainly concentrated in A 3 -A 4 Cheng Tianlin's team inserted TadA deaminase into the middle of dUn1Cas12f1-RRA (D143R, T147R, E151A) and mutated TadA deaminase to develop Un1Cas12f1-derived miniABEs and miniCBEs. Ma Hanhui's team fused the transcriptional activation element VPR to the C-terminus of dUn1Cas12f1-3R (D143R, T147R, T203R) to develop dUn1Cas12f1-3R-VPR. In order to further improve the efficiency of transcriptional activation, they fused the double-stranded DNA binding domain Sso7d to the C-terminus of dUn1Cas12f1-3R-VPR to construct Sso7d-dUn1Cas12f1-3R-VPR-C (SminiCRa), further increasing the activation level by about 4 times. In addition to transcription activation tools, Ma Hanhui's team also replaced VPR with KRAB to develop transcription inhibition tools miniCRi (dUn1Cas12f1-3R-KRAB) and SminiCRi (dUn1Cas12f1-3R-KRAB-Sso7d).

[0006] However, the above research did not solve the limitation of Cas12f's narrow targeting range, and the editing efficiency of optimized Cas12f nucleases and derived editing tools in mammalian cells and animals is still lower than that of traditional large nucleases SpCas9 and AsCas12a. Therefore, it is necessary to develop Cas12f gene editing tools with higher activity and wider targeting range. Summary of the invention

[0008] The main purpose of the present invention is to provide a Cas12f nuclease with a wider targeting range and a gene editing tool derived therefrom to solve the defect of the existing Cas12f editing tools with a narrow targeting range.

[0009] The present invention provides a Cas12f nuclease with a wider targeting range, including single point mutations or combined mutations of all amino acid residues in the Un1Cas12f1 nuclease; wherein the single point mutation Un1Cas12f1 is: D143S, L152S, Q244R, N133S, E151V, K129N, G130R, N133C, Q244F, P247Q, S135Y, V137C, H139Y, T203M, S246F, Y140W, G200E, G130Q, S135K, V144C, I158M, E20 6F, E206Q, Q242T, S246A, I131K, S168C, G200R, Q201P, Y202M, K248S, I131R, A134D, S136V, E138Q, R148W, G199F, A134Q, S136P, V137N, Y140A, C145S, R148V, E151W, L152H, K154S, K167H, G199Y, Y202L, K245H, K248I; the combined mutant Un1Cas12f1 is any combination of the above single point mutations.

[0010] More preferably, the combined mutant Un1Cas12f1 is a combination of 2-5 of the above-mentioned single-point mutants.

[0011] Most preferably, the combined mutant Un1Cas12f1 is: D143S / L152S / Q244R / N133S / E151V, named Cas12f-SSRSV, and its sequence is shown in SEQ NO.1.

[0012] Furthermore, the Cas12f nuclease also includes other mutations or other modifications to perform precise editing functions.

[0013] Preferably, the Cas12f nuclease further includes a Q197K mutation or a mutation at the same position, which is recorded as Cas12f-SSRSVK, and its sequence is shown in SEQ NO. 2. According to conventional knowledge in the art, the Q197 residue can be mutated into other amino acids to achieve editing.

[0014] The present invention also provides an adenine base editor comprising the Cas12f nuclease.

[0015] The present invention also provides a cytosine base editor comprising the Cas12f nuclease.

[0016] The present invention also provides a transcription activation tool comprising the Cas12f nuclease.

[0017] Preferably, the above-mentioned nuclease, adenine base editor, cytosine base editor or transcription activation tool all include a promoter, and the promoter is selected from at least one of CMV, CAG, PGK, EF1α, Ctsk (muscle-specific promoter), and Lp (liver-specific promoter).

[0018] Preferably, the above-mentioned nuclease, adenine base editor, cytosine base editor or transcription activation tool all include a nuclear localization signal NLS, and the NLS is selected from SV40 NLS or bpNLS, or other types of nuclear localization signals.

[0019] Preferably, the above-mentioned nuclease, adenine base editor, cytosine base editor or transcription activation tool all include polyadenylic acid polyA, and the polyA selects bovine growth hormone polyadenylation signal BGH polyA, or other species include eukaryotic prokaryotic polyadenylation signals.

[0020] Preferably, the linker used by the adenine base editor, cytosine base editor or transcriptional activation tool to connect MCP and effector protein is (SGGS) 2 -XTEN-(SGGS) 2 linker, or other types of linkers.

[0021] Preferably, the above-mentioned adenine base editor includes the deaminase TadA8e (F148A), and the deaminase TadA selects the Escherichia coli-derived deaminase TadA or its mutant, as well as other deaminases that can achieve A to G mutation.

[0022] Preferably, the above-mentioned adenine base editor is SSRSV-ABE, composed of CMV-SV40 NLS-dUn1Cas12f1(SSRSV)-bpNLS-P2A-SV40 NLS-MCP-32aa linker-TadA8e(F148A)-bpNLS-BGH polyA, and its sequence is shown in SEQ NO.3, and also includes other permutations and combinations, including other Cas12f nucleases with DNA recognition and cutting functions, and also includes other position changes such as adenine deaminase embedded in the middle of the nuclease.

[0023] Most preferably, the sgRNA of the above-mentioned adenine base editor SSRSV-ABE contains an MS2 hairpin structure, and the MS2 hairpin structure is inserted in Stem loop 4, and its sequence is shown in SEQ NO.6. It also includes inserting the MS2 hairpin structure into other positions, inserting different numbers of MS2 hairpin structures, or replacing it with a hairpin structure that is compatible with other RNA binding proteins.

[0024] Preferably, the above-mentioned cytosine base editor includes deaminase A3A (D131P), and the deaminase A3A selects human APOBEC3A or its mutants, as well as other deaminases that can achieve C to T mutation.

[0025] Preferably, the above-mentioned cytosine base editor is SSRSV-CBE, composed of CMV-SV40 NLS-dUn1Cas12f1(SSRSV)-bpNLS-P2A-SV40 NLS-MCP-32aa linker-A3A(D131P)-bpNLS-T2A-2xUGI-SV40NLS-BGH polyA, and its sequence is shown in SEQ NO.4, and also includes other permutations and combinations, including other Cas12f nucleases with DNA recognition and cutting functions, and also including other position changes such as cytosine deaminase embedded in the middle of the nuclease.

[0026] Most preferably, the sgRNA of the above-mentioned cytosine base editor SSRSV-CBE contains an MS2 hairpin structure, and the MS2 hairpin structure is inserted in Stem loop 4, and its sequence is shown in SEQ NO.6. It also includes inserting the MS2 hairpin structure into other positions, inserting different numbers of MS2 hairpin structures, or replacing it with a hairpin structure compatible with other RNA binding proteins.

[0027] Preferably, the above-mentioned transcriptional activation tool comprises a transcriptional activation element, which is P65-HSF1 or a mutant thereof, and other transcriptional activation elements capable of activating gene expression.

[0028] Preferably, the above-mentioned transcription activation tool is PHM-SSRSV, composed of CMV-SV40 NLS-P65-HSF1-32aalinker-MCP-bpNLS-P2A-SV40 NLS-dUn1Cas12f1 (SSRSV)-bp NLS-BGH polyA, and its sequence is shown in SEQ NO.5, and also includes other permutations and combinations, and also includes other Cas12f nucleases with DNA recognition and cutting functions, and also includes other position changes such as transcription activation elements embedded in the middle of the nuclease.

[0029] Most preferably, the sgRNA of the above-mentioned adenine base editor SSRSV-ABE contains an MS2 hairpin structure, and the MS2 hairpin structure is inserted at the 5' end and Stem loop 2, and its sequence is shown in SEQ NO.7. It also includes inserting the MS2 hairpin structure into other positions, inserting different numbers of MS2 hairpin structures, or replacing it with a hairpin structure compatible with other RNA binding proteins.

[0030] The present invention also provides an application of a Cas12f nuclease with a wider targeting range, an adenine base editor, a cytosine base editor or a transcription activation tool in gene editing.

[0031] Preferably, the nuclease, adenine base editor, cytosine base editor achieves base editing by plasmid transfection; or by transfection of mRNA, electroporation of protein; or by delivery of DNA, mRNA or protein by nanoliposomes, viruses and viroids.

[0032] Preferably, the cells of the nuclease, adenine base editor, cytosine base editor or transcription activation tool are HEK293T cells; or other cells derived from humans and other mammals; or all kinds of animal and plant cells; or other gametes and fertilized eggs derived from humans and other mammals.

[0033] Preferably, the cells used are eukaryotic cells, but also include non-eukaryotic cells, such as prokaryotes, ancient organisms, etc. It also includes editing, treatment, gene expression regulation, etc. that can be achieved in animals.

[0034] Preferably, the gene editing includes gene therapy, cell therapy, animal model construction and crop genetic breeding.

[0035] Beneficial Effects

[0036] The present invention screens out 71 point mutations that can cut NTTR and TNTR PAM through a bacterial forward screening system, and further screens on the endogenous targets of NTTR and TNTR PAM, and finally screens out five key point mutations of D143S, L152S, Q244R, N133S, and E151V, and successfully develops Un1Cas12f1 mutant Cas12f-SSRSV with a wider targeting range, and evaluates its editing characteristics; at the same time, the present invention constructs adenine base editor SSRSV-ABE, cytosine base editor SSRSV-CBE and transcription activation tool PHM-SSRSV with a wider targeting range through the MS2-MCP strategy. Compared with other Cas12f-related ABEs, SSRSV-ABE has a wider targeting range, higher editing efficiency, and fewer bystander editing. It is a precise adenine base editor with higher PAM compatibility. Compared with miniCBE, SSRSV-CBE greatly improves editing efficiency while broadening the targeting range. PHM-SSRSV can improve the transcription level of endogenous gene mRNA at the NTNR PAM endogenous target. The high PAM compatibility and high activity of Cas12f-SSRSV, SSRSV-ABE, SSRSV-CBE and PHM-SSRSV provide more options for target design. The design of the present invention is derived from the actual needs of gene editing tools in clinical transformation, and small nucleases, ABE, CBE and transcription activation tools with higher PAM compatibility are developed, which have certain clinical transformation value. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the forward screening principle of Un1Cas12f1 library.

[0039] Figure 2 Forward screening of Un1Cas12f1 mutants that recognize NTTR and TNTR PAMs for bacteria.

[0040] Figure 3 Screening for Un1Cas12f1 mutants that recognize NTTR and TNTR PAMs for endogenous targets.

[0041] Figure 4 The editing efficiency of Cas12f-SSRSV at the NTTR and TNTR PAM endogenous targets.

[0042] Figure 5 Cas12f-SSRSV has a preference for the second base of PAM.

[0043] Figure 6 Schematic diagram of the principle of GFP expression activated cell line.

[0044] Figure 7 PAM preference of wild-type Un1Cas12f1 and Cas12f-SSRSV.

[0045] Figure 8 The editing efficiency of Cas12f-SSRSV at the NTNR PAM endogenous target site.

[0046] Fig. 9 Schematic diagram of the partial crystal structure of Un1Cas12f1 (PDB: 7C7L).

[0047] Fig.10 Screening of Cas12f-SSRSV mutants for the VVTR PAM endogenous target.

[0048] Fig.11 is the preference of Cas12f-SSRSVK for the second base of PAM.

[0049] Fig.12 This is a schematic diagram of the MCP-MINI ABE structure.

[0050] Fig.13 The effect of different MCP-MINI ABE structures on editing efficiency.

[0051] Fig.14 Insert schematic for MS2 tagging.

[0052] Fig.15 The effects of different MS2 tag insertion positions and numbers on editing efficiency.

[0053] Fig.16 The effect of linker length between MCP and TadA8e on editing efficiency.

[0054] Fig.17 Effects of different TadA8e mutants on the editing window.

[0055] Fig.18 The editing efficiency of SSRSV-ABE at the endogenous target site of 293T.

[0056] Fig.19 Comparison of the editing efficiency of four Cas12f-ABEs.

[0057] Fig. 20 Comparison of the editing windows of four Cas12f-ABEs.

[0058] Fig.21 Comparison of editing efficiency between miniCBE and SSRSV-CBE.

[0059] Fig. 22 Schematic diagram of the principle of GFP transcriptional activation cell line.

[0060] Fig.23 Optimization of Cas12f-MPH sgRNA structure.

[0061] Fig.24 Schematic diagram of the Cas12f-MPH structure.

[0062] Fig.25 Optimization of the Cas12f-MPH structure.

[0063] Fig.26 The transcriptional activation level of endogenous genes of PHM-SSRSV in HEK293T. DETAILED DESCRIPTION

[0065] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0066] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present invention.

[0067] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions.

[0068] Example 1

[0069] Based on the crystal structure of Un1Cas12f1 (PDB: 7C7L), 59 amino acids (K129-S168, K196-I207, Q242-K248) close to the PAM double strand were selected to construct a single-site saturation mutation library, and a bacterial forward screening system suitable for Un1Cas12f1 was established ( Figure 1 ). 71 point mutations that can cut NTTR and TNTRPAM were screened out through the bacterial forward screening system ( Figure 2 ), and further screened on the endogenous targets of NTTR and TNTR PAM ( Figure 3 ), and finally screened out five key point mutations, D143S, L152S, Q244R, N133S, and E151V. These five point mutations were superimposed together to form Cas12f-SSRSV, whose sequence is shown in SEQ NO.1.

[0070] The editing efficiency of Cas12f-SSRSV was tested on 24 endogenous targets of NTTR and TNTR PAM ( Figure 4 ), the results showed that the editing efficiency of wild-type Un1Cas12f1 was between 2.26% and 53%, and the editing efficiency of Cas12f-SSRSV was between 14.79% and 71.41%, which was 5.2 times higher than that of wild-type Un1Cas12f1. In general, by combining bacterial forward screening with endogenous target screening, a mutant Un1Cas12f1, Cas12f-SSRSV, was developed that can recognize and cut NTTR and TNTR PAM.

[0071] We tested whether Cas12f-SSRSV could recognize and cleave the endogenous target of NNTR PAM ( Figure 5 ), among the 40 endogenous targets of NNTRPAM, the editing efficiency of Cas12f-SSRSV was increased by an average of 15.4 times compared with the wild-type Un1Cas12f1. It was also found that Cas12f-SSRSV may have a certain preference for the second position T.

[0072] So we activated the cell line by expressing GFP ( Figure 6 ) analyzed the PAM of Cas12f-SSRSV. This cell line inserted a 5bp random sequence as the PAM of Cas12f after the GFP start codon ATG, and inserted a 24bp target sequence after the random sequence. Since the sum of the inserted PAM sequence and the target sequence is not an integer multiple of 3, GFP is not expressed. However, when transferred into a plasmid expressing Cas12f and sgRNA, if Cas12f recognizes PAM and cuts the target sequence, resulting in insertions or deletions, it may restore the GFP reading frame and activate GFP expression. By sorting out GFP-positive cells for deep sequencing, the PAM recognized by Cas12f can be detected. The results showed that ( Figure 7 ), the PAM of the wild-type Un1Cas12f1 is TTTR, and T at the second position is very conservative. Although Cas12f-SSRSV greatly reduces the restriction of Un1Cas12f1 on the second position T, there is still a certain preference. However, Cas12f-SSRSV has almost no restrictions on the first and third positions of PAM. Therefore, the PAM of Cas12f-SSRSV is defined as NTNR.

[0073] The editing efficiency of Cas12f-SSRSV was tested at 40 endogenous target sites of NTNR PAM ( Figure 8 ), the results showed that the editing efficiency of Cas12f-SSRSV was increased by an average of 9.5 times compared with the wild-type Un1Cas12f1.

[0074] Cas12f-SSRSV was further optimized based on the crystal structure, attempting to eliminate the restriction on the second T position of PAM ( Fig. 9 ). We selected two amino acid residues, Y146 and Q197, that interact with the second T·A base pair at the TTTR PAM, and subjected these two amino acids to single-point saturation mutations. The mutations were introduced into Cas12f-SSRSV and screened at the endogenous target site of VVTR PAM ( Fig.10 ). It was found that after the introduction of the Q197K mutation, the editing efficiency of Cas12f-SSRSV was greatly improved at four of the six screened targets, so the mutant was named Cas12f-SSRSVK, and its sequence is shown in SEQ NO.2. After testing a large number of NNTR PAM endogenous targets, it was found that the editing efficiency of Cas12f-SSRSVK at the NCTR PAM target was greatly improved ( Fig.11 ), but the editing efficiency at the NTTR PAM target is lower than that of Cas12f-SSRSV. Therefore, it is believed that Cas12f-SSRSVK does not eliminate the restriction on the second T of PAM, but prefers the NCTR PAM target.

[0075] Example 2

[0076] Based on the MS2 tag-MCP RNA binding protein strategy, we developed Cas12f-related adenine base editors SSRSV-ABE and cytosine base editors SSRSV-CBE with a wider targeting range. First, the MS2 tag was fused to the 5' end of the sgRNA, and MCP and TadA8e were fused to the N-terminus or C-terminus of dUn1Cas12f1 in different orders to construct four MCP-MINI ABEs ( Fig.12), TadA8e or MCP and dUn1Cas12f1 are connected by a P2A self-cleaving peptide. When the protein is expressed, the ribosome skips the formation of the peptide bond between the glycine and proline at the P2AC end, but does not affect the subsequent protein translation, thereby translating the proteins at the N-terminus and C-terminus of P2A separately to form two independent proteins. The test was carried out on the HEK293T endogenous target sites of three TTTR PAMs (Figure 13), and the optimal structure was dUn1Cas12f1-P2A-MCP-TadA8e (C1). Next, the insertion position of the MS2 tag was optimized, and the MS2 tag was inserted into different stem loops of the sgRNA (Figure 14). The test results showed that the optimal sgRNA structure was S4-MS2 ( Fig.15 ). The length of the linker between MCP and TadA8e was also optimized (Figure 16). The results showed that linkers of different lengths had no effect on the editing efficiency, but C1 still had a high A-to-G editing efficiency at the far end of PAM, so point mutations were introduced in TadA8e to narrow the editing window. Tests were performed on the endogenous targets of three TTTR PAMs in HEK293T (Figure 17). The introduction of F148A not only narrowed the editing window but also improved the editing efficiency. The CMV-SV40 NLS-dUn1Cas12f1-bpNLS-P2A-SV40NLS-MCP-32aa linker-TadA8e(F148A)-bpNLS-BGH polyA was named MCP-MINI ABE.

[0077] Example 3

[0078] SSRSV mutations were introduced into MCP-MINI ABE to construct SSRSV-ABE, whose sequence is shown in SEQ NO.3, and tested at the endogenous target site in HEK293T ( Fig.18 ), the average A-to-G editing efficiency of SSRSSV-ABE was 17.83% at 12 non-TTTR PAM targets and 28.83% at 5 TTTR PAM targets, which were 9.73 times and 1.26 times higher than those of MCP-MINI ABE, respectively. Compared with the Cas12f-ABE systems dCasMINI-ABE, TaRGET-C3.1, and miniABE reported in previous articles, SSRSV-ABE has a higher editing efficiency, with an average editing efficiency of 25.3% at the 6 tested targets, and a narrower editing window, mainly concentrated in A 3 , A 4 Bit( Fig.19 , Fig. 20 ).

[0079] The TadA in SSRSV-ABE was replaced with cytosine deaminase A3A-D131P and UGI was added to develop SSRSV-CBE, whose sequence is shown in SEQ NO.4. It was tested on the endogenous target and found that ( Fig.21 ), compared with the previously reported miniCBE, SSRSV-CBE further improved the editing efficiency while broadening the PAM.

[0080] Example 4

[0081] The same MS2-MCP strategy was used to develop the Cas12f-MPH transcriptional activation system. First, a GFP transcriptional activation cell line was constructed ( Fig. 22 ), insert 7 repeats of TTTG PAM and the target sequence in front of the TRE3G promoter. If dUn1Cas12f1 binds to the target sequence and recruits MCP-p65-HSF1 to the vicinity of the TRE3G promoter through the MS2 tag on the sgRNA, it will activate the transcription and expression of GFP. The transcriptional activation efficiency of Cas12f-MPH can be determined by detecting the proportion of GFP-positive cells through flow cytometry.

[0082] According to the optimal structure C1 of SSRSV-ABE, a transcriptional activation system was constructed. MCP-p65-HSF1 was connected to the C-terminus of dUn1Cas12f1 through the P2A self-cleaving peptide. The MS2 tag was inserted into the 5' end of sgRNA or different stem loops, and the number of MS2 tags was increased to detect the transcriptional activation efficiency of Cas12f-MPH. The test results showed that the optimal sgRNA structure was 5'-MS2+S2-MS2 ( Fig.23 ). Next, we will continue to optimize the structure of Cas12f-MPH ( Fig.24 ), a total of four work systems were constructed for testing, and the results showed that ( Fig.25 )CMV-SV40 NLS-P65-HSF1-32aa linker-MCP-bpNLS-P2A-SV40 NLS-dUn1Cas12f1-bp NLS-BGH polyA is the optimal transcription activation system, which is named PHM-Cas12f.

[0083] The SSRSV point mutation was introduced into PHM-Cas12f to construct CMV-SV40 NLS-P65-HSF1-32aa linker-MCP-bpNLS-P2A-SV40 NLS-dUn1Cas12f1(SSRSV)-bp NLS-BGH polyA (hereinafter referred to as PHM-SSRSV), whose sequence is shown in SEQ NO.5, in an attempt to expand the targeting range of PHM-Cas12f, and tested on 6 HEK293T endogenous target sites. The results are shown in Fig.26 As shown. The transcriptional activation levels of PHM-SSRSV on all six targets were higher than those of PHM-Cas12f. Compared with untreated cells, the relative expression levels of target gene mRNA in cells treated with PHM-SSRSV increased by 66-4734 times, and the relative expression levels of target gene mRNA in cells treated with PHM-Cas12f increased by 3.85-681 times. The above results show that compared with PHM-Cas12f, PHM-SSRSV further improves the transcriptional activation level while broadening the targeting range.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Cas12f nuclease with a wider targeting range, characterized in that: Including all single point mutations or combined mutations of amino acid residues in Un1Cas12f1 nuclease; wherein the single point mutation Un1Cas12f1 is: D143S, L152S, Q244R, N133S, E151V, K129N, G130R, N133C, Q244F, P247Q, S135Y, V137C, H139Y, T203M, S246F, Y140W, G200E, G130Q, S135K, V144C, I158M, E206F, E206Q, Q242 T, S246A, I131K, S168C, G200R, Q201P, Y202M, K248S, I131R, A134D, S136V, E138Q, R148W, G199F, A134Q, S136P, V137N, Y140A, C145S, R148V, E151W, L152H, K154S, K167H, G199Y, Y202L, K245H, K248I; the combined mutant Un1Cas12f1 is any combination of the above single point mutations.

2. The Cas12f nuclease according to claim 1, characterized in that: The combined mutant Un1Cas12f1 is a combination of 2-5 single-point mutants.

3. The Cas12f nuclease according to claim 1 or 2, characterized in that: The combined mutant Un1Cas12f1 is: D143S / L152S / Q244R / N133S / E151V.

4. The Cas12f nuclease according to claim 1, characterized in that: The Cas12f nuclease also includes other mutations or other modifications to perform precise editing functions.

5. The Cas12f nuclease according to claim 1 or 4, characterized in that: The Cas12f nuclease also includes a Q197K mutation or a mutation at the same position.

6. An adenine base editor comprising the Cas12f nuclease of claim 1.

7. A cytosine base editor comprising the Cas12f nuclease of claim 1.

8. A transcriptional activation tool comprising the Cas12f nuclease of claim 1.

9. The nuclease, adenine base editor, cytosine base editor or transcription activation tool according to any one of claims 1 to 8, characterized in that: They all include a promoter, and the promoter is selected from at least one of CMV, CAG, PGK, EF1α, Ctsk, and Lp1.

10. The nuclease, adenine base editor, cytosine base editor or transcription activation tool according to any one of claims 1 to 8, characterized in that: All of them include a nuclear localization signal NLS, and the NLS is selected from SV40 NLS or bpNLS, or other types of nuclear localization signals.

11. The nuclease, adenine base editor, cytosine base editor or transcriptional activation tool according to any one of claims 1 to 8, characterized in that: All include polyadenylic acid polyA, wherein the polyA is selected from bovine growth hormone polyadenylation signal BGH polyA, or other types of polyadenylation signals.

12. The adenine base editor, cytosine base editor or transcription activation tool according to any one of claims 6 to 8, characterized in that: All of them contain RNA binding protein, and the RNA binding protein is MS2 bacteriophage coat protein MCP, or other types of RNA binding protein.

13. The adenine base editor, cytosine base editor or transcription activation tool according to any one of claims 6 to 8, characterized in that: The linker used to connect MCP and effector protein is (SGGS)2-XTEN-(SGGS)2, with a length of 32 amino acids, or other types of linkers.

14. An adenine base editor according to claim 6, characterized in that: The invention comprises deaminase TadA8e (F148A), wherein the deaminase TadA is selected from deaminase TadA derived from Escherichia coli or its mutant, and other deaminases capable of achieving A to G mutation.

15. An adenine base editor according to claim 6, characterized in that: The connection sequence between adenine deaminase, dCas12f, MCP, nuclear localization signal and linker that constitute ABE is arranged in different orders.

16. An adenine base editor according to claim 6, characterized in that: ABE sgRNAs all contain an MS2 hairpin structure, which is inserted into Stem loop 4, or inserted into other positions, or inserted in different numbers, or adapted to other types of RNA binding proteins.

17. The cytosine base editor according to claim 7, characterized in that: It includes deaminase A3A (D131P), wherein the deaminase A3A selects human APOBEC3A or its mutant, and other deaminases that can achieve C to T mutation.

18. The cytosine base editor according to claim 7, characterized in that: The connection sequence between cytosine deaminase, UGI, dCas12f, MCP, nuclear localization signal and linker that constitute CBE is arranged in different orders.

19. The cytosine base editor according to claim 7, characterized in that: CBE sgRNAs all contain an MS2 hairpin structure, which is inserted in Stem loop 4, or in other positions, or in different numbers, or is compatible with other types of RNA-binding proteins.

20. The transcriptional activation means according to claim 8, characterized in that: The invention comprises a transcriptional activation element, wherein the transcriptional activation element is P65-HSF1 or its mutant, and other transcriptional activation elements capable of realizing gene expression activation.

21. The transcriptional activation means according to claim 8, characterized in that: The connection sequence between the transcription activation element P65, the transcription activation element HSF1, dCas12f, MCP, the nuclear localization signal and the linker that constitute the transcription activation tool is arranged in a different order.

22. The transcriptional activation means according to claim 8, characterized in that: ABE sgRNAs all contain an MS2 hairpin structure, which is inserted at the 5' end and in Stem loop 2, or at other locations, or in different numbers, or adapted to other types of RNA binding proteins.

23. A use of a nuclease, adenine base editor, cytosine base editor or transcription activation tool as described in any one of claims 1 to 8 in gene editing.

24. The use according to claim 23, characterized in that: The cells used to test the nuclease, adenine base editor, cytosine base editor or transcription activation tool are HEK293T cells; or other cells derived from humans and other mammals; or all kinds of animal and plant cells; or other gametes and fertilized eggs derived from humans and other mammals.

25. The use according to claim 24, characterized in that: The cell is a eukaryotic cell gene editing; or a non-eukaryotic cell gene editing; or an editing that can be achieved in an animal.

26. The use according to any one of claims 23 to 25, characterized in that: The gene editing includes gene therapy, cell therapy, animal model construction and crop genetic breeding.

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