A tigr-tas-based gene editing system and application thereof

Abstract

This invention relates to a TIGR-Tas-based gene editing system and its applications, belonging to the field of biotechnology. This invention develops a genome editing tool based on the TIGR-Tas system to overcome the PAM / TAM dependency problem prevalent in existing gene editing tools, thereby broadening the editable scope in the *B. subtilis* genome. First, this invention verifies the editing activity of different Tas nucleases in *B. subtilis*, creating a TAM / PAM-free gene editing system. Based on this, the single-target editing activity of the system is further improved by optimizing the TigRNA array and expression system, and its multi-target editing capability is evaluated. Finally, the TIGR-Tas system is coupled with NHEJ pathways from different sources to create a highly efficient gene editing platform that is PAM-free and HDR-free, providing a novel tool for achieving efficient gene editing in *B. subtilis*.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a gene editing system based on TIGR-Tas and its applications. Background Technology

[0002] RNA-guided systems are multifunctional molecular tools that precisely locate functional proteins to target sites through base complementarity between guide RNA and target gene sequences, mediating specific interactions between functional proteins and different target genes. Compared with traditional protein-guided editing technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), RNA-guided systems offer multiple advantages, including ease of design, traceability, and programmability. Based on these characteristics, the CRISPR-Cas system has been developed into a universal genome editing tool, enabling targeted gene knockout, knock-in, and point mutation in various hosts, and has further spawned various tools such as the CRISPR transcriptional regulation system (CRISPRi / a), base editing systems (BE), and prime editing systems (PE).

[0003] However, the large molecular size of classic Cas proteins such as Cas9 and Cas12a limits the flexibility of editing plasmid construction and the transformation efficiency in non-model chassis. Compact RNA-guided systems such as the OMEGA system (TnpB, IscB) and the Fanzor system, discovered in recent years, have been developed into diverse gene editing tools and have shown editing activity comparable to Cas9 in some hosts. However, the targeting recognition function of these systems depends on specific PAM or TAM sequences, resulting in a limited number of editable sites in the genome, thus limiting the applicability and flexibility of RNA-guided editing tools. Although a series of PAM-relaxed variants can be obtained through directed evolution of nucleases, expanding the targeting range of existing tools, the editing activity of these variants often decreases accordingly. Figure 1 ).

[0004] Existing genome editing tools in Bacillus subtilis face multiple limitations. First, commonly used RNA-guided nucleases (such as Cas9 and Cas12a) generally rely on specific PAM sequences, limiting the operational range of gene editing tools on the genome. Second, HDR-based genome editing processes depend on long donors and highly efficient recombinant proteins, but the efficiency of the B. subtilis endogenous homologous recombination repair system is insufficient, making multi-target editing difficult to achieve efficiently. Furthermore, the large molecular size of traditional Cas proteins further increases the difficulty of plasmid construction and reduces transformation efficiency. Although compact nucleases (such as the OMEGA system) that have emerged in recent years have partially alleviated these burdens, they still do not escape PAM / TAM dependence, and their editing activity and stability in B. subtilis have not been fully validated. Therefore, there is a need to develop a tool that is PAM-independent, has a compact protein structure, and can achieve efficient multi-target editing in B. subtilis. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem of PAM dependence in the genome editing and derivative tools commonly used in the prior art by RNA-guided nucleases.

[0006] To address the aforementioned technical problems, this invention provides a TIGR-Tas-based gene editing system and its applications. This invention develops a genome editing tool based on the TIGR-Tas system to overcome the PAM / TAM dependency problem prevalent in existing gene editing tools, thereby broadening the editable scope in the *B. subtilis* genome. First, this invention verifies the editing activity of different Tas nucleases in *B. subtilis*, creating a TAM / PAM-free gene editing system. Based on this, the single-target editing activity of the system is further improved by optimizing the TigRNA array and expression system, and its multi-target editing capability is evaluated. Finally, the TIGR-Tas system is coupled with NHEJ pathways from different sources to create a highly efficient gene editing platform that is PAM-free and HDR-free, providing a novel tool for achieving efficient gene editing in *B. subtilis*.

[0007] The first objective of this invention is to provide a TIGR-Tas-based gene editing system, which includes a nuclease and a TigRNA, wherein the nuclease is selected from TasH or TasR nucleases, and the TigRNA includes a marginal repeat sequence, a spacer sequence A, a circular repeat sequence, a spacer sequence B, and a terminal repeat sequence. The spacer sequence A and spacer sequence B are complementary to portions of the DNA strand of the target gene, and the marginal repeat sequence, the circular repeat sequence, and the terminal repeat sequence together guide the nuclease to target and cleave the target gene.

[0008] Furthermore, when the nuclease is a TasR nuclease, the marginal repeat sequence is AGCCA, the circular repeat sequence is TGAAAACCC, and the terminal repeat sequence is TGCG. When the nuclease is a TasH nuclease, the marginal repeat sequence is AGTCA, the circular repeat sequence is AGACAACCA, and the terminal repeat sequence is AGCG.

[0009] Further, the amino acid sequence of the TasH nuclease is shown in SEQ ID NO.1, the amino acid sequence of the Ta-TasR nuclease is shown in SEQ ID NO.2, and the amino acid sequence of the Par-TasR nuclease is shown in SEQ ID NO.3.

[0010] Furthermore, the nuclease is processed via P tet The promoter initiates expression, and the TigRNA is expressed by P. tet promoter or P veg Promoter startup expression.

[0011] Furthermore, the P tet The nucleotide sequence of the promoter is shown in SEQ ID NO.11, P veg The nucleotide sequence of the promoter is shown in SEQ ID NO.12.

[0012] Furthermore, the host bacteria of the gene editing system includes Bacillus subtilis.

[0013] A second objective of this invention is to provide an application of the above-described gene editing system in the preparation of gene editing products.

[0014] Furthermore, the gene editing product also includes a non-homologous end ligation repair system, wherein the non-homologous end ligation repair system comprises Ku protein and LigD ligase.

[0015] Further, the amino acid sequence of the Ku protein is shown in SEQ ID NO.4 or SEQ ID NO.6, and the amino acid sequence of the LigD ligase is shown in SEQ ID NO.5 or SEQ ID NO.7.

[0016] A third objective of this invention is to provide a gene editing method for Bacillus subtilis, wherein the above-mentioned gene editing system is transferred into a host bacterium, and the host bacterium is Bacillus subtilis.

[0017] Furthermore, the host bacteria also include a non-homologous end-joint repair system.

[0018] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0019] This invention validated the editing activities of different Tas nucleases in *B. subtilis*, creating a TAM / PAM-free gene editing system. Based on this, the single-target editing activity of the system was further improved by optimizing the TigRNA array and expression system, and its multi-target editing capability was evaluated. Finally, the TIGR-Tas system was coupled with NHEJ pathways from different sources to create a highly efficient gene editing platform that is PAM-free and HDR-free, providing a novel tool for achieving efficient gene editing in *B. subtilis*. Attached Figure Description

[0020] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0021] Figure 1 This section discusses the current application status of RNA-guided nucleases and the advantages of TIGR-Tas.

[0022] Figure 2 The advantages and mechanisms of action of the NHEJ repair pathway;

[0023] Figure 3 It is based on the design, construction, and optimization of the TIGR-Tas editing system;

[0024] Figure 4 This is an evaluation of the multi-target editing activity of the TIGR-Tas editing system;

[0025] Figure 5 It is a targeted knockout mediated by the TIGR-Tas system coupled with the NHEJ pathway. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0027] DNA polymerase was purchased from Nanjing Novizan Biotechnology Co., Ltd., seamless cloning kit from Ibotek Biotechnology Co., Ltd., and plasmid extraction kit and PCR product nucleic acid purification kit from Magen Biotechnology Co., Ltd. Restriction endonucleases and T4 ligases were purchased from NEB.

[0028] LB medium (for plasmid construction and cell culture) mainly contains NaCl (10 g / L), tryptone (10 g / L), and yeast extract (5 g / L). The antibiotics used during culture mainly include kanamycin (50 mg / L), ampicillin (100 mg / L), chloramphenicol (5 mg / L), acyclovir (1 mM / L) at a final concentration, and IPTG (0.5 mM / L) at a final concentration.

[0029] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, cell biology, recombinant DNA technology, and related fields, which have been well described in existing literature.

[0030] The TIGR-Tas system is a novel RNA-guided nuclease system independent of CRISPR. Its effector protein is called Tas nuclease, and its guide RNA is called TigRNA. Figure 1 This system combines the advantages of compact protein structure and multi-target RNA array processing activity, providing a structural and functional basis for the development of miniature multiplex multi-target gene editing tools. Unlike traditional CRISPR systems, each unit in the tandemly arranged TigRNA array contains spacer sequences (spacer A and spacer B), which can pair with the two complementary strands of the target DNA, thereby achieving precise recognition and binding to the target DNA. This unique dual-spacer sequence-mediated targeting mechanism frees the TIGR system from dependence on PAM or TAM sequences, broadening the targetable range of existing RNA-guided editing tools.

[0031] Example 1: Design, Construction, and Optimization of an Editing System Based on TIGR-Tas

[0032] To construct a TIGR gene editing tool applicable to *B. subtilis*, this study synthesized three TIGR nucleases (TasH, Ta-TasR, and Par-TasR) based on the codon bias of *B. subtilis*, and designed corresponding TigRNA sequences for each (Table 1). Based on these, corresponding single-plasmid gene editing systems were constructed. In each system, both the TIGR nuclease and the TigRNA were derived from Par-TasR. tet Promoter-induced expression ( Figure 3 (A) Using sfgfp integrated into the genome as the editing target, the knockout efficiency of each system was evaluated. The results showed that the editing efficiencies of TasH, Ta-TasR, and Par-TasR were 28.40%, 14.10%, and 17.66%, respectively. Figure 3 (B in the middle).

[0033] Table 1. TigRNAs used to validate TIGR-Tas editing activity.

[0034]

[0035] To improve the gene editing efficiency of the system, the promoter expressing TigRNA was replaced with a strong constitutive promoter P. veg Furthermore, the effect of TigRNA GC content on editing activity was investigated. Results showed that in P... veg Under conditions that drive TigRNA expression, the knockout efficiency of Ta-TasR and Par-TasR is improved compared to induced expression, while the efficiency of the TasH system does not change significantly. Figure 3 (C in the text). Further systematic adjustment of the GC content of TigRNA revealed that it had limited impact on the editing activities of Ta-TasR and TasH, but Par-TasR could achieve 100% knockout efficiency at a GC content of around 40%, indicating that Par-TasR has a preference for the GC content of TigRNA. Figure 3 (C in the middle).

[0036] Example 2: Evaluation of TIGR-Tas multi-target editing activity

[0037] To evaluate the multi-target editing capability of the TIGR-Tas system, this study designed corresponding TigRNAs and knockout verification primers (Tables 2 and 3) for different target gene combinations (sfgfp(G) and aprE(A), nprE(N), or epr(E)). Dual-target knockout experiments were performed using Par-TasR and Ta-TasR, respectively. The results showed that Par-TasR only produced transformants targeting the sfgfp and epr combination, with 100% of colonies showing no fluorescence. Colony PCR confirmed that both targets were successfully knocked out. Figure 4 (A in the text). The Ta-TasR system produced transformants in both sfgfp and nprE and the sfgfp and epr combination, with non-fluorescent colony percentages of 26.58% and 50.53%, respectively. PCR validation showed that all non-fluorescent colonies achieved complete knockout of the corresponding target gene. Figure 4 (A in the middle).

[0038] Table 2. TigRNA sequences used to evaluate TIGR-Tas multi-target editing activity.

[0039]

[0040] Table 3 Primer Sequences

[0041]

[0042] When performing simultaneous knockout of three targets, Par-TasR only produced transformants in the combination of targeting sfgfp, aprE, and epr, with a fluorescent colony rate of 13.22%. Colony PCR validation showed that two genes were knocked out simultaneously, but no simultaneous knockout of all three genes was detected. Figure 4 (B in the text). Similarly, using Ta-TasR, transformants grew in both the simultaneous knockout of sfgfp, aprE, and nprE combinations and the sfgfp, nprE, and epr combination, with non-fluorescent colonies accounting for 38.42% and 9.80%, respectively. Colony PCR verification only detected co-knockout of two genes, failing to achieve simultaneous knockout of all three targets. Figure 4 (B in the middle).

[0043] These results indicate that both Par-TasR and Ta-TasR possess multi-target array processing activity and can mediate simultaneous knockout of two genes, but their editing efficiency is significantly affected by the target combination. Par-TasR exhibits more efficient editing activity under specific target combinations. However, in the three-target simultaneous knockout experiments, only co-knockout of two genes was observed; no simultaneous knockout of all three genes was detected. This suggests that endogenous HDR activity may be a limiting factor in more complex multi-target editing in B. subtilis, making efficient multiple editing difficult.

[0044] Example 3: TIGR-Tas coupled with NHEJ pathway-mediated targeted knockout

[0045] To address the limitations of the HDR pathway in multiple editing, this study further explored the application potential of coupling TIGR-Tas with NHEJ. Four different NHEJ components were selected for the experiment: the endogenous NHEJ pathway from *B. subtilis* 168 – BsNHEJ (BsKu sequence as shown in SEQ ID NO.4, BsligD sequence as shown in SEQ ID NO.5), the NHEJ pathway from *M. smegmatis* – MsmNHEJ (MsmKu sequence as shown in SEQ ID NO.6, MsmligD sequence as shown in SEQ ID NO.7), the NHEJ pathway from *M. tuberculosis* – MtbNHEJ (MtbKu sequence as shown in SEQ ID NO.8, MtbLigD sequence as shown in SEQ ID NO.9), and the bacterial phage-derived (T4-LigD) (T4 ligase sequence as shown in SEQ ID NO.10). In all editing systems, the Par-TasR nuclease, along with its corresponding Ku protein and LigD ligase, is induced to express by the Ptet promoter. Figure 5 (A in the middle).

[0046] Using the Par-NHEJ editing system to mediate sfgfp knockout experiments, the results showed that effective editing and repair could only be achieved when Par-TasR was coupled with the NHEJ pathway (BsNHEJ) derived from B. subtilis. Specifically, when coupled with BsNHEJ, the proportion of non-fluorescent colonies reached approximately 50%-60%. Figure 5 (B in the text). PCR and sequencing verification were performed on non-fluorescent colonies. The results showed that the electrophoretic bands were of varying sizes. Combined with the sequencing results, this further confirmed that random-sized fragment insertions and deletions occurred near the target site during the editing and repair process. Figure 5 (B and C in the text).

[0047] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A gene editing system based on TIGR-Tas, characterized in that, The gene editing system includes a nuclease and TigRNA, wherein the nuclease is a TasH nuclease or a TasR nuclease, and the TigRNA includes a marginal repeat sequence, a spacer sequence A, a circular repeat sequence, a spacer sequence B, and a terminal repeat sequence. The spacer sequence A and the spacer sequence B are complementary to portions of the DNA strand of the target gene, respectively. The marginal repeat sequence, the circular repeat sequence, and the terminal repeat sequence together guide the nuclease to target and cleave the target gene.

2. The gene editing system according to claim 1, characterized in that, The amino acid sequence of the TasH nuclease is shown in SEQ ID NO.1; The TasR nuclease is either a Ta-TasR nuclease or a Par-TasR nuclease. The amino acid sequence of the Ta-TasR nuclease is shown in SEQ ID NO.2, and the amino acid sequence of the Par-TasR nuclease is shown in SEQ ID NO.

3.

3. The gene editing system according to claim 1, characterized in that, The nuclease is expressed from P tet The TigRNA is expressed from P tet The nuclease is expressed from P veg The TigRNA is expressed from P 4. The gene editing system according to claim 1, characterized in that, When the nuclease is TasH nuclease, the marginal repeat sequence is AGTCA, the circular repeat sequence is AGACAACCA, and the terminal repeat sequence is AGCG; When the nuclease is TasR nuclease, the marginal repeat sequence is AGCCA, the circular repeat sequence is TGAAACCC, and the terminal repeat sequence is TGCG.

5. The gene editing system according to claim 1, characterized in that, The host bacteria of the gene editing system include Bacillus subtilis.

6. The use of the gene editing system according to any one of claims 1-5 in the preparation of gene editing products.

7. The gene editing product according to claim 6, characterized in that, The gene editing product also includes a non-homologous end ligation repair system, wherein the non-homologous end ligation repair system comprises Ku protein and LigD ligase.

8. The gene-editing product according to claim 7, characterized in that, The amino acid sequence of the Ku protein is shown in SEQ ID NO.4 or SEQ ID NO.6, and the amino acid sequence of the LigD ligase is shown in SEQ ID NO.5 or SEQ ID NO.

7.

9. A gene editing method for Bacillus subtilis, characterized in that, The method involves converting the gene editing system described in any one of claims 1-5 into a host bacterium, wherein the host bacterium is Bacillus subtilis.

10. The Bacillus subtilis gene editing method according to claim 9, characterized in that, The host bacteria also include a non-homologous end-joint repair system.

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