Cas12i3-based fusion protein and application thereof in mediating gene editing

Abstract

The invention relates to a fusion protein based on Cas12i3 and application of the fusion protein in mediating gene editing, the fusion protein is formed by fusing NLSSV 40, Cas12i3, NLS, IEE and XTEN, the amino acid sequence of the NLSSV 40 is shown as SEQ ID No. 3, the amino acid sequence of the Cas12i3 is shown as SEQ ID No. 5, the amino acid sequence of the NLS is shown as SEQ ID No. 7, the amino acid sequence of the IEE is shown as SEQ ID No. 9, and the amino acid sequence of the XTEN is shown as SEQ ID No. 11.

Description

Technical Field

[0001] The present invention relates to the field of functional proteins, and in particular to fusion proteins based on Cas12i3 and applications thereof in mediating gene editing. Background Art

[0002] With the continuous development of biotechnology, gene editing technology plays an increasingly important role in plant gene function research and crop genetic improvement. Among them, the CRISPR / Cas system has become one of the most widely used gene editing tools due to its high efficiency and convenience.

[0003] As a member of the CRISPR / Cas system, Cas12i3 has unique gene editing properties. It can recognize specific DNA sequences and cut the target DNA through its endonuclease activity. Compared with some commonly used gene editing systems such as Cas9, Cas12i has unique advantages, such as recognizing different PAM sequences, which increases its selection range of editable sites in plant genomes. But overall, its editing efficiency is still a certain distance away from some mature gene editing systems. Therefore, it is necessary to further improve and optimize the CRISPR / Cas12i3 gene editing system, improve its editing efficiency, and develop a more efficient gene editing system. Summary of the invention

[0004] One of the present invention provides a fusion protein, which consists of NLS SV40 , Cas12i3, NLS, IEE and XTEN are fused, wherein the NLS SV40 The amino acid sequence of is shown in SEQ ID No. 3, the amino acid sequence of Cas12i3 is shown in SEQ ID No. 5, the amino acid sequence of NLS is shown in SEQ ID No. 7, the amino acid sequence of IEE is shown in SEQ ID No. 9, and the amino acid sequence of XTEN is shown in SEQ ID No. 11.

[0005] In one embodiment, the fusion protein is composed of IEE, XTEN, NLS SV40 , Cas12i3, NLS, XTEN and IEE are connected in series from N-terminus to C-terminus.

[0006] In one embodiment, the fusion protein is composed of IEE, XTEN, NLS SV40 , Cas12i3, and NLS are connected in series from the N-terminus to the C-terminus.

[0007] In one embodiment, the fusion protein is composed of NLS SV40, Cas12i3, NLS, XTEN and IEE are connected in series from N-terminus to C-terminus.

[0008] The second aspect of the present invention provides a nucleic acid encoding a fusion protein as described in any one of the first aspects of the present invention.

[0009] In one embodiment, the NLS SV40 The nucleic acid sequence of is shown as SEQ ID No. 2, the nucleic acid sequence of Cas12i3 is shown as SEQ ID No. 4, the nucleic acid sequence of NLS is shown as SEQ ID No. 6, the nucleic acid sequence of IEE is shown as SEQ ID No. 8, and the nucleic acid sequence of XTEN is shown as SEQ ID No. 10.

[0010] The third aspect of the present invention provides a gene editing system, which includes a first regulatory element and a second regulatory element; wherein the first regulatory element is the nucleic acid described in the second aspect of the present invention, and the first regulatory element encodes a fusion protein as described in any one of the first aspect of the present invention; the second regulatory element includes a crRNA expression box for gene editing.

[0011] In a specific embodiment, the nucleic acid sequence of the promoter used by the first regulatory element is shown as SEQ ID No. 16 or SEQ ID No. 18.

[0012] In a specific embodiment, the nucleic acid sequence of the terminator used in the first regulatory element is SEQ ID No. 19.

[0013] In a specific embodiment, the nucleic acid sequence of kozak used in the first regulatory element is shown as SEQ ID No.1.

[0014] In a specific embodiment, the crRNA expression cassette comprises a promoter, an expression element and a terminator, wherein the nucleic acid sequence of the expression element is shown in SEQ ID No. 17.

[0015] In a specific embodiment, the nucleic acid sequence of the promoter used for the crRNA expression cassette is shown as SEQ ID No. 16 or SEQ ID No. 18.

[0016] In a specific embodiment, the nucleic acid sequence of the terminator used for the crRNA expression cassette is SEQ ID No. 19.

[0017] The fourth aspect of the present invention provides the use of the fusion protein described in any one of the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention, or the gene editing system described in the third aspect of the present invention in deletion editing.

[0018] In a specific embodiment, the application is for editing DNA in the genome of a Poaceae plant; In one embodiment, the grass plant is rice.

[0019] Beneficial effects of the present invention: The fusion protein of the present invention improves the deletion editing efficiency of the CRISPR / Cas12i3 system. DETAILED DESCRIPTION

[0020] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.

[0021] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels. Example 1

[0022] Optimizing kozak-NLS based on rice codons SV40 -Cas12i3-NLS tandem sequence (i.e. kozak, SV40 first nuclear localization signal, Cas12i3 and second nuclear localization signal are connected in series from 5' to 3' end), commissioned Beijing Qingke Biotechnology Co., Ltd. to be synthesized and cloned into pUC57 vector to obtain pUC57-Cas12i3. BamHI and BcuI were used to double-digest pUC57-Cas12i3 and pUbi-Cas9NG respectively, and then the double-digested target fragments were connected, that is, Cas9NG in pUbi-Cas9NG was replaced with the above kozak-NLS SV40- Cas12i3-NLS tandem sequence, to obtain the vector pUbi-Cas12i3. Among them, the kozak sequence is shown in SEQ ID No. 1; SV40 first nuclear localization signal NLS SV40 The base sequence is shown in SEQ ID No. 2, and the base sequence encoded by it is shown in SEQ ID No. 3; the base sequence of Cas12i3 is shown in SEQ ID No. 4, and the amino acid sequence encoded by it is shown in SEQ ID No. 5; the base sequence of the second nuclear localization signal NLS is shown in SEQ ID No. 6, and the base sequence encoded by it is shown in SEQ ID No. 7.

[0023] The base sequence of the IEE protein fused with the Cas12i3 protein for improving editing efficiency (IEE) is shown in SEQ ID No. 8, and the amino acid sequence encoded by it is shown in SEQ ID No. 9; the base sequence of the adapter sequence XTEN is shown in SEQ ID No. 10, and the amino acid sequence encoded by it is shown in SEQ ID No. 11. The two IEEs were connected together using two XTEN adapter sequences, and an EcoRV restriction site was set between the two XTENs, and a kozak sequence (as shown in SEQ ID No. 1) was fused to the 5' of the first IEE, i.e., kozak-IEE-XTEN-EcoRV-XTEN-IEE. The entire coding region of kozak-IEE-XTEN-EcoRV-XTEN-IEE was entrusted to Beijing Qingke Biotechnology Co., Ltd. for artificial synthesis and cloned into the pUC57 vector to obtain pUC57-IEE. pUC57-IEE was digested with EcoRV and then dephosphorylated to obtain a dephosphorylated pUC57-IEE linear fragment. The NLS was amplified by high-fidelity PCR using plasmid pUbi-Cas12i3 as a template and Cas12i3-F1 (SEQ ID No. 12) and Cas12i3-R1 (SEQ ID No. 13) as primers. SV40 -Cas12i3-NLS 3252bp, dephosphorylated pUC57-IEE linear fragment and NLS SV40 -Cas12i3-NLS PCR fragment Infusion, construct the vector pUC57-IEE-Cas12i3-IEE. Then use BamHI and BcuI to double-digest pUC57-IEE-Cas12i3-IEE and pUbi-Cas9NG, respectively, and then connect the double-digested target fragments, that is, replace Cas9NG in pUbi-Cas9NG with the above kozak-IEE-XTEN-NLS SV40 -Cas12i3-NLS-XTEN- IEE tandem sequence to obtain the vector pUbi-IEE-Cas12i3-IEE.

[0024] For the construction of pUbi-IEE-Cas12i3 and pUbi-Cas12i3-IEE vectors, the plasmid pUC57-IEE was double-digested with EcoRV / BcuI or BamHI / EcoRV to remove the XTEN-IEE sequence of 976 bp at the 3' end or the kozak-IEE-XTEN sequence of 986 bp at the 5' end, respectively, to obtain the pUC57-kozak-IEE-XTEN linear fragment or the pUC57-XTEN-IEE linear fragment; the NLS was amplified by high-fidelity PCR using the plasmid pUC57-Cas12i3 as a template and Cas12i3-F1 and Cas12i3-R2 (SEQ ID No. 14) as a primer pair. SV40 -Cas12i3-NLS PCR product, pUC57-kozak-IEE-XTEN linear fragment and NLS SV40 -Cas12i3-NLS PCR fragment was infused and connected to obtain the vector pUC57-IEE-Cas12i3; plasmid pUC57-Cas12i3 was used as a template, Cas12i3-F2 (SEQ ID No. 15) and Cas12i3-R1 were used as primer pairs, and kozak-NLS was obtained by high-fidelity enzyme PCR amplification. SV40 -Cas12i3-NLS PCR product, pUC57-XTEN-IEE linear fragment and kozak-NLS SV40 -Cas12i3-NLS PCR fragment was infused and connected to pUC57-Cas12i3-IEE. Then, pUC57-IEE-Cas12i3 and pUbi-Cas9NG were double-digested with BamHI and BcuI, respectively, and then the double-digested target fragments were connected, that is, Cas9NG in pUbi-Cas9NG was replaced with the above kozak-IEE-XTEN-NLS SV40 -Cas12i3-NLS tandem sequence to obtain the vector pUbi-IEE-Cas12i3; double-digest pUC57-Cas12i3-IEE and pUbi-Cas9NG with BamHI and BcuI, respectively, and then connect the double-digested target fragments, that is, replace Cas9NG in pUbi-Cas9NG with the above-mentioned kozak-NLS SV40 -Cas12i3-NLS-XTEN-IEE concatenated sequence to obtain vector pUbi-Cas12i3-IEE.

[0025] The above kozak-IEE-XTEN-NLS SV40-Cas12i3-NLS-XTEN-IEE tandem sequence, kozak-IEE-XTEN-NLS SV40 -Cas12i3-NLS and Kozak-NLS SV40 The promoters used in the Cas12i3-NLS-XTEN-IEE tandem sequences are all Ubi promoters (SEQ ID No. 16).

[0026] Beijing Qingke Biotechnology Co., Ltd. was commissioned to artificially synthesize tRNA-DR-crRNA-BsaI-BsaI-DR (SEQ ID No. 17) and cloned into a vector containing U6 promoter (SEQ ID No. 18) and tNOS terminator (SEQ ID No. 19), named pHZ33. Among them, BsaI restriction enzyme digestion can insert a 23bp spacer for targeting. Example 2

[0027] The transcript and genomic sequences of the genes to be edited were obtained from the MSU / TIGR Rice Genome Database (http: / / rice.plantbiology.msu.edu / ). The TTN PAM motif sequence was searched in the 3'ʹ direction, where N is A or G or C or T, and the spacer sequence was synthesized and inserted into the pHZ33 vector.

[0028] For the OsCPK18 (Os07g0409900) gene, the spacer sequence used for targeting is shown in SEQ ID No. 20, and the primers are fgCPK18-F (SEQ ID No. 21, acac matching the BsaI restriction site is introduced at the 5' end) and fgCPK18-R (SEQ ID No. 22, ctct matching the BsaI restriction site is introduced at the 5' end). According to the spacer sequence of the gene, specific PCR primers for identification were designed: HT-gOsCPK18-F (SEQ ID No. 23) and HT-gOsCPK18-R (SEQ ID No. 24).

[0029] After synthesizing the primers, fgCPK18-F and fgCPK18-R were phosphorylated using T4 polynucleotide kinase and annealed to form double strands. The double strands formed by fgCPK18-F and fgCPK18-R were then cloned into the BsaI restriction site of the pHZ33 vector. The sequencing results showed that they were correct, and pHZ33-fgCPK18 was obtained, in which the forward primer represented the spacer sequence inserted into the restriction site on the pHZ33 vector. pHZ33-fgCPK18 was constructed with pUbi-Cas12i3, pUbi-IEE-Cas12i3-IEE, pUbi-IEE-Cas12i3, and pUbi-Cas12i3-IEE through Gateway's LR reaction to form the final rice genetic transformation vectors pUbi-Cas12i3-HZ33-fgCPK18, pUbi-IEE-Cas12i3-IEE-HZ33-fgCPK18, pUbi-IEE- Cas12i3-HZ33-fgCPK18, and pUbi-Cas12i3-IEE-HZ33-fgCPK18.

[0030] pUbi-Cas12i3-HZ33-fgCPK18, pUbi-IEE-Cas12i3-IEE-HZ33-fgCPK18, pUbi-IEE-Cas12i3-HZ33-fgCPK18 and pUbi-Cas12i3-IEE-HZ33-fgCPK18 were transformed into japonica rice variety NG46 using Agrobacterium EHA105 (EHA105 electroporated competent cells were purchased from Beijing Bomade Gene Technology Co., Ltd.), and then DNA was extracted from T0 generation regenerated rice plants using the CTAB method. Genomic DNA of T0 generation regenerated rice plants was used as a template, PCR amplification was performed using HT-gOsCPK18-F and HT-gOsCPK18-R as primer pairs, and the PCR products were sequenced and detected. After analysis and sequencing, the editing sequence of OsCPK18 was obtained. The results showed that each vector produced deletion editing of the bases inside or near the target sequence (spacer sequence) in the rice genome. Among them, the editing efficiency of pUbi-Cas12i3-HZ33-fgCPK18 was 22.2%. The editing efficiencies of pUbi-IEE-Cas12i3-IEE-HZ33-fgCPK18, pUbi-IEE- Cas12i3-HZ33-fgCPK18, and pUbi-Cas12i3-IEE-HZ33-fgCPK18 connected with IEE were 56.3%, 97.9%, and 95.1%, respectively, which were significantly improved compared with the editing efficiency of pUbi-Cas12i3-HZ33-fgCPK18. Example 3

[0031] For the OsCPK19 (Os07g0515100) gene, the spacer sequence used for targeting is shown in SEQ ID No. 25, and the primers are fgCPK19-F (SEQ ID No. 26, acac matching the BsaI restriction site is introduced at the 5' end) and fgCPK19-R (SEQ ID No. 27, ctct matching the BsaI restriction site is introduced at the 5' end). According to the spacer sequence of the gene, specific PCR primers for identification are designed: HT-gOsCPK19-F (SEQ ID No. 28) and HT-gOsCPK19-R (SEQ ID No. 29). The forward primer represents the spacer sequence inserted into the restriction site on the pHZ33 vector.

[0032] The other operations were the same as in Example 2, and pUbi-Cas12i3-HZ33-fgCPK19, pUbi-IEE-Cas12i3-IEE-HZ33-fgCPK19, pUbi-IEE-Cas12i3-HZ33-fgCPK19 and pUbi-Cas12i3-IEE-HZ33-fgCPK19 were finally obtained, and they were transformed into japonica rice variety NG46 by Agrobacterium EHA105 transformation method, and then DNA was extracted from T0 generation regenerated rice plants by CTAB method. Using the genomic DNA of T0 generation regenerated rice plants as a template, PCR amplification was performed with HT-gOsCPK19-F and HT-gOsCPK19-R as primer pairs, and the PCR products were sequenced and detected. After analysis and sequencing, the editing sequence of OsCPK19 was obtained. The results showed that each vector produced deletion editing of the bases inside or near the target sequence (spacer sequence) in the rice genome. Among them, the editing efficiency of pUbi-Cas12i3-HZ33-fgCPK19 was 34%, and the editing efficiencies of pUbi-IEE-Cas12i3-IEE-HZ33-fgCPK19, pUbi-IEE- Cas12i3-HZ33-fgCPK19, and pUbi-Cas12i3-IEE-HZ33-fgCPK19 connected with IEE were 30%, 95.8%, and 88.9%, respectively. It can be seen that the editing efficiency of pUbi-IEE-Cas12i3-HZ33-fgCPK19 and pUbi-Cas12i3-IEE-HZ33-fgCPK19 is greatly improved compared with pUbi-Cas12i3-HZ33-fgCPK19. Example 4

[0033] For the OsCPK20 (Os07g0568600) gene, the spacer sequence used for targeting is shown in SEQ ID No. 30, and the primers are fgCPK20-F (SEQ ID No. 31, acac matching the BsaI restriction site is introduced at the 5' end) and fgCPK20-R (SEQ ID No. 32, ctct matching the BsaI restriction site is introduced at the 5' end). According to the spacer sequence of the gene, specific PCR primers for identification are designed: HT-gOsCPK20-F (SEQ ID No. 33) and HT-gOsCPK20-R (SEQ ID No. 34). The reverse primer represents the spacer sequence inserted into the restriction site on the pHZ33 vector.

[0034] Other operations were the same as in Example 2. Finally, pUbi-Cas12i3-HZ33-fgCPK20, pUbi-IEE-Cas12i3-IEE-HZ33-fgCPK20, pUbi-IEE-Cas12i3-HZ33-fgCPK20 and pUbi-Cas12i3-IEE-HZ33-fgCPK20 were transformed into japonica rice variety NG46 by Agrobacterium EHA105 transformation method, and then DNA was extracted from T0 generation regenerated rice plants by CTAB method. Using the genomic DNA of T0 generation regenerated rice plants as a template, PCR amplification was performed with HT-gOsCPK20-F and HT-gOsCPK20-R as primer pairs, and the PCR products were sequenced and detected. After analysis and sequencing, the editing sequence of OsCPK20 was obtained. The results showed that each vector produced deletion editing of the bases inside or near the target sequence (spacer sequence) in the rice genome. Among them, the editing efficiency of pUbi-Cas12i3-HZ33-fgCPK20 was 20.8%. The editing efficiencies of pUbi-IEE-Cas12i3-IEE-HZ33-fgCPK20, pUbi-IEE- Cas12i3-HZ33-fgCPK20, and pUbi-Cas12i3-IEE-HZ33-fgCPK20 connected with IEE were 50%, 90.7%, and 89.13%, respectively, which were significantly improved compared with the editing efficiency of pUbi-Cas12i3-HZ33-fgCPK20.

Claims

1. A fusion protein consisting of NLS SV40 , Cas12i3, NLS, IEE and XTEN are fused, among which, The NLS SV40 The amino acid sequence of , is shown in SEQ ID No. 3, the amino acid sequence of Cas12i3 is shown in SEQ ID No. 5, the amino acid sequence of NLS is shown in SEQ ID No. 7, the amino acid sequence of IEE is shown in SEQ ID No. 9, and the amino acid sequence of XTEN is shown in SEQ ID No.

11.

2. The fusion protein according to claim 1, characterized in that The fusion protein is composed of IEE, XTEN, NLS SV40 , Cas12i3, NLS, XTEN and IEE are connected in series from N-terminus to C-terminus.

3. The fusion protein according to claim 1, characterized in that The fusion protein is composed of IEE, XTEN, NLS SV40 , Cas12i3, and NLS are connected in series from the N-terminus to the C-terminus.

4. The fusion protein according to claim 1, characterized in that The fusion protein consists of NLS SV40 , Cas12i3, NLS, XTEN and IEE are connected in series from N-terminus to C-terminus.

5. A nucleic acid encoding the fusion protein according to any one of claims 1 to 4.

6. The nucleic acid according to claim 5, characterized in that The NLS SV40 The nucleic acid sequence of is shown as SEQ ID No. 2, the nucleic acid sequence of Cas12i3 is shown as SEQ ID No. 4, the nucleic acid sequence of NLS is shown as SEQ ID No. 6, the nucleic acid sequence of IEE is shown as SEQ ID No. 8, and the nucleic acid sequence of XTEN is shown as SEQ ID No.

10.

7. A gene editing system comprising a first regulatory element and a second regulatory element; wherein: The first regulatory element is the nucleic acid of claim 5 or 6, and the first regulatory element encodes the fusion protein of any one of claims 1 to 4; The second regulatory element includes a crRNA expression box for gene editing.

8. The gene editing system according to claim 7, characterized in that: The crRNA expression cassette comprises a promoter, an expression element and a terminator, wherein the nucleic acid sequence of the expression element is shown in SEQ ID No.

17.

9. Use of the fusion protein described in any one of claims 1 to 4, the nucleic acid described in claim 5 or 6, or the gene editing system described in claim 7 or 8 in deletion editing.

10. The use according to claim 9, characterized in that: The application is to edit DNA in the genome of Gramineae plants; Preferably, the grass plant is rice.