Construction of a New Mini Genome Editing System Based on Csy4 and MCP

doi:10.13560/j.cnki.biotech.bull.1985.2023-0248

Abstract

Abstract:

MCP is the coat protein of MS2 phage, and Csy4 is a small protein involved in crRNA generation of CRISPR 1-F system, which can recognize and bind RNA with high specificity. CRISPR/Cas and other genome editing technologies have some problems, such as large molecular weight of targeted nuclease, high off-target rate, and limited by PAM site. To solve these problems and construct new mini genome editing system based on Csy4 and MCP, AlphaFold2 was used to predict the structures of MCP-FokI, FokI-MCP, Csy4-FokI and FokI-Csy4 fusion proteins. The MCP-FokI and FokI-MCP editing vectors were transformed into Arabidopsis thaliana by the flower dip method, respectively, and the CLCrV-mediated Csy4-FokI and FokI-Csy4 editing systems were delivered using A. thaliana leaf injection. The A. thaliana genomic DNA was extracted and the editing ability of the novel mini-genome editing system was tested by HI-TOM high-throughput sequencing. The results showed that each domain of MCP, FokI and Csy4 maintained their original three-dimensional structures in the fusion proteins, suggesting that they all functioned properly. The plant expression vectors of MCP-FokI and MCP-FokI binding dual target sites with four different intermediate spacer regions were constructed to target the AtCLA1 genes. Sequencing results demonstrated that neither MCP-FokI nor FokI-MCP achieved the targeted editing of the target genes. Seven CLCrV-mediated Csy4-FokI and FokI-Csy4 editing vectors with different intermediate spacer regions were constructed to target the AtCLA1 genes. Among them the CLCrV-mediated Csy4-FokI editing system was able to achieve targeted editing of the target gene, but the mutation types were all base substitution types and the editing efficiency was low. No genome editing was detected in the FokI-Csy4 system. Taken together, a novel Csy4-FokI mini-genome editing system was successfully constructed, which may provide an innovative solution to overcome the problems of CRISPR/Cas genome editing technology.

Key words: Csy4, MCP, cotton leaf crumple virus, VIGE

DENG Jia-hui, LEI Jian-feng, ZHAO Yi, LIU Min, HU Zi-yao, YOU Yang-zi, SHAO Wu-kui, LIU Jian-fei, LIU Xiao-dong. Construction of a New Mini Genome Editing System Based on Csy4 and MCP[J]. Biotechnology Bulletin, 2023, 39(10): 68-79.

Figures/Tables 8

Fig. 1 Schematic diagram of MCP-FokI（FokI-MCP）and Csy4-FokI（FokI-Csy4）genome editing system A: Overview of MS2-SL-guided FokI nuclease principle: Two MCP-FokI fusion proteins recruit two different MS2-SLs to the target gene locus, promoting Fok I cleavage of the double-stranded DNA. B: Overview of sgRNA-guided FokI nuclease principle: Two Csy4-FokI fusion proteins recruit two different sgRNAs to the target gene locus, promoting Fok I cleavage of the double-stranded DNA

Table 1 Primers and applications

引物Primer	序列Sequence（5'-3'）	用途Application
FokI-Link-Csy4-F	CCTCCGGGGGATCTTCAGGAGGATCAATGGGAGATCATTATCTTGATATTAG	构建FokI-Csy4 Construction of FokI-Csy4
FokI-Link-Csy4-R	GCCTTTTTCGTGGCCGCCGGCCTTTTGAACCAAGGAACAAAACCTC	构建FokI-Csy4 Construction of FokI-Csy4
T-Csy4-FokI-F	GGTCGGTATCCACGGAGTCCCAGCAGCCATGGGAGATCATTATCTTGATATTAG	构建Csy4-FokI Construction of Csy4-FokI
T-Csy4-FokI-R	GACGATCCGCCGGAGGACCCTCCAGAGAACCAAGGAACAAAACCTC	构建Csy4-FokI Construction of Csy4-FokI
T-sgRNA1-F	TAGAGTCGACATAGCGATTGGTCATTAAGAATCACAATCGTTCACTGCCGTATAGGC	构建AtU6∷sgRNA1 Construction of AtU6∷sgRNA1
T-sgRNA1-R	CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG	构建AtU6∷sgRNA1 Construction of AtU6∷sgRNA1
T-sgRNA2-F	GAGTCGACATAGCGATTGAATCACAATCATATCAGAGGTTCACTGCCGTATAGGC	构建AtU6∷sgRNA2 Construction of AtU6∷sgRNA2
T-sgRNA2-R	CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG	构建AtU6∷sgRNA2 Construction of AtU6∷sgRNA2
T-sgRNA3-F	CTAGAGTCGACATAGCGATTGAAGAATCACAATCATATCAGTTCACTGCCGTATAGGC	构建AtU6∷sgRNA3 Construction of AtU6∷sgRNA3
T-sgRNA3-R	CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG	构建AtU6∷sgRNA3 Construction of AtU6∷sgRNA3
T-sgRNA4-F	GAGTCGACATAGCGATTGAGAATCACAATCATATCAGGTTCACTGCCGTATAGGC	构建AtU6∷sgRNA4 Construction of AtU6∷sgRNA4
T-sgRNA4-R	CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG	构建AtU6∷sgRNA4 Construction of AtU6∷sgRNA4
T-sgRNA5-F	GTCGAAGTAGTGATTGGCTTATGAAGCCATGAACAGTTCACTGCCGTATAGGC	构建AtU6-26∷sgRNA5 Construction of AtU6-26∷sgRNA5
T-sgRNA5-R	TTGTCGACGCCCGGGATCCGGATCTCGAGTAAGAGCTCG	构建AtU6-26∷sgRNA5 Construction of AtU6-26∷sgRNA5
T-sgRNA6-F	GTAGTGATTGGAAGCCATGAACAACGCCGGTTCACTGCCGTATAGGC	构建AtU6-26∷sgRNA6 Construction of AtU6-26∷sgRNA6
T-sgRNA6-R	TTGTCGACGCCCGGGATCCGGATCTCGAGTAAGAGCTCG	构建AtU6-26∷sgRNA6 Construction of AtU6-26∷sgRNA6
KP-U6-SK-F	GGTACCTTAATTAAGGAGTGATCAAAAGTCCCAC	构建Kpn I-Pac I-AtU6∷sgRNA（1 2 3 4）-EcoR I Construction of Kpn I-Pac I-AtU6∷sgRNA（1 2 3 4）-EcoR I
E-U6-SK-R	GAATTCGATCCGGATCTCGAGTAAGA
E-U6-26-SK-F	GAATTCGAATTGCCCTTAAGCTTCGTTG	构建EcoR I-AtU6-26∷sgRNA（5 6）-Xba I-Avr II Construction of EcoR I-AtU6-26∷sgRNA（5 6）-Xba I-Avr II
XM-U6-26-SK-R	TCTAGACCTAGGGATCCGGATCTCGAGTAAGA
AT-FC/CF-F	GACCACCAACTCCATTACTTG	PCR扩增AtCLA1基因涵盖靶位点区域 The PCR amplification is performed to target and amplify the specific region of the AtCLA1
AT-FC/CF-R	GAGAGCCGGGTTAGACTGTA
Spe I-CLF-1F	ACTAGTTGCCTGCAGGTCAACATG	构建Spe I-FokI-Csy4（Csy4-FokI）-Pac I Construction of Spe I-FokI-Csy4（Csy4- FokI）-Pac I
Pac I-CLF-1r	TTAATTAATAGTAACATAGATGACACCGC
HiT-CLA1-F	GGAGTGAGTACGGTGTGCGGTTGCTGTGATTGGTGATGG	高通量扩增AtCLA1基因涵盖靶位点区域 High-throughput amplification is performed to cover the target region of the AtCLA1
HiT-CLA1-R	GAGTTGGATGCTGGATGGTCCATCCAAAGTAGCTGTAGGT
CLCrVA-F	GCTGCTTGTATGTTTGGGTG	CLCrV-A 病毒检测 CLCrV-A virus detection
CLCrVA-R	CAAGGGAGAGTAGTTGGCA	CLCrV-A 病毒检测 CLCrV-A virus detection

Table 1 Primers and applications

引物Primer	序列Sequence（5'-3'）	用途Application
FokI-Link-Csy4-F	CCTCCGGGGGATCTTCAGGAGGATCAATGGGAGATCATTATCTTGATATTAG	构建FokI-Csy4 Construction of FokI-Csy4
FokI-Link-Csy4-R	GCCTTTTTCGTGGCCGCCGGCCTTTTGAACCAAGGAACAAAACCTC	构建FokI-Csy4 Construction of FokI-Csy4
T-Csy4-FokI-F	GGTCGGTATCCACGGAGTCCCAGCAGCCATGGGAGATCATTATCTTGATATTAG	构建Csy4-FokI Construction of Csy4-FokI
T-Csy4-FokI-R	GACGATCCGCCGGAGGACCCTCCAGAGAACCAAGGAACAAAACCTC	构建Csy4-FokI Construction of Csy4-FokI
T-sgRNA1-F	TAGAGTCGACATAGCGATTGGTCATTAAGAATCACAATCGTTCACTGCCGTATAGGC	构建AtU6∷sgRNA1 Construction of AtU6∷sgRNA1
T-sgRNA1-R	CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG	构建AtU6∷sgRNA1 Construction of AtU6∷sgRNA1
T-sgRNA2-F	GAGTCGACATAGCGATTGAATCACAATCATATCAGAGGTTCACTGCCGTATAGGC	构建AtU6∷sgRNA2 Construction of AtU6∷sgRNA2
T-sgRNA2-R	CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG	构建AtU6∷sgRNA2 Construction of AtU6∷sgRNA2
T-sgRNA3-F	CTAGAGTCGACATAGCGATTGAAGAATCACAATCATATCAGTTCACTGCCGTATAGGC	构建AtU6∷sgRNA3 Construction of AtU6∷sgRNA3
T-sgRNA3-R	CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG	构建AtU6∷sgRNA3 Construction of AtU6∷sgRNA3
T-sgRNA4-F	GAGTCGACATAGCGATTGAGAATCACAATCATATCAGGTTCACTGCCGTATAGGC	构建AtU6∷sgRNA4 Construction of AtU6∷sgRNA4
T-sgRNA4-R	CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG	构建AtU6∷sgRNA4 Construction of AtU6∷sgRNA4
T-sgRNA5-F	GTCGAAGTAGTGATTGGCTTATGAAGCCATGAACAGTTCACTGCCGTATAGGC	构建AtU6-26∷sgRNA5 Construction of AtU6-26∷sgRNA5
T-sgRNA5-R	TTGTCGACGCCCGGGATCCGGATCTCGAGTAAGAGCTCG	构建AtU6-26∷sgRNA5 Construction of AtU6-26∷sgRNA5
T-sgRNA6-F	GTAGTGATTGGAAGCCATGAACAACGCCGGTTCACTGCCGTATAGGC	构建AtU6-26∷sgRNA6 Construction of AtU6-26∷sgRNA6
T-sgRNA6-R	TTGTCGACGCCCGGGATCCGGATCTCGAGTAAGAGCTCG	构建AtU6-26∷sgRNA6 Construction of AtU6-26∷sgRNA6
KP-U6-SK-F	GGTACCTTAATTAAGGAGTGATCAAAAGTCCCAC	构建Kpn I-Pac I-AtU6∷sgRNA（1 2 3 4）-EcoR I Construction of Kpn I-Pac I-AtU6∷sgRNA（1 2 3 4）-EcoR I
E-U6-SK-R	GAATTCGATCCGGATCTCGAGTAAGA
E-U6-26-SK-F	GAATTCGAATTGCCCTTAAGCTTCGTTG	构建EcoR I-AtU6-26∷sgRNA（5 6）-Xba I-Avr II Construction of EcoR I-AtU6-26∷sgRNA（5 6）-Xba I-Avr II
XM-U6-26-SK-R	TCTAGACCTAGGGATCCGGATCTCGAGTAAGA
AT-FC/CF-F	GACCACCAACTCCATTACTTG	PCR扩增AtCLA1基因涵盖靶位点区域 The PCR amplification is performed to target and amplify the specific region of the AtCLA1
AT-FC/CF-R	GAGAGCCGGGTTAGACTGTA
Spe I-CLF-1F	ACTAGTTGCCTGCAGGTCAACATG	构建Spe I-FokI-Csy4（Csy4-FokI）-Pac I Construction of Spe I-FokI-Csy4（Csy4- FokI）-Pac I
Pac I-CLF-1r	TTAATTAATAGTAACATAGATGACACCGC
HiT-CLA1-F	GGAGTGAGTACGGTGTGCGGTTGCTGTGATTGGTGATGG	高通量扩增AtCLA1基因涵盖靶位点区域 High-throughput amplification is performed to cover the target region of the AtCLA1
HiT-CLA1-R	GAGTTGGATGCTGGATGGTCCATCCAAAGTAGCTGTAGGT
CLCrVA-F	GCTGCTTGTATGTTTGGGTG	CLCrV-A 病毒检测 CLCrV-A virus detection
CLCrVA-R	CAAGGGAGAGTAGTTGGCA	CLCrV-A 病毒检测 CLCrV-A virus detection

Fig. 2 Tertiary structure prediction of fusion protein A: Prediction diagram of FokI-MCP and MCP-FokI fusion protein structure.B: Structure prediction of FokI-Csy4 and Csy4-FokI fusion protein

Fig. 3 Construction of MCP-FokI and FokI-MCP gene editing vector M: 2 K Plus II DNA standard molecular weight. WT: Wild type. A: AtCLA1 as target, left side-SL and right side-SL transcribed with AtU6-26 and AtU6 promoters respectively for the same target. B: Schematic diagram of MCP-FokI and FokI-MCP editing vector construction. C: MCP-FokI and FokI-MCP editing vector enzymatic identification. D: Enzymatic identification of four different intermediate spacer target sequences. E: Enzymatic identification

Fig. 4 Targeted knockout of AtCLA1 gene by MCP-FokI and FokI-MCP gene editing system The red arrow indicates transgenic resistant seedlings

Fig. 5 Construction of Csy4-FokI and FokI-Csy4 gene editing vector M: Standard molecular weight of 2K Plus II DNA. WT: Wild type. A: Transcription of left sgRNA and right sgRNA with AtU6-26 and AtU6 promoters respectively for the same target site using AtCLA1 as the target. B: Schematic diagram of Csy4-FokI and Csy4-FokI editing vector construction. C: Csy4-FokI- Bzro and FokI-Csy4-B zro editing vector enzymatic identification results. D: Enzymatic identification results of seven different intermediate spacer region target sequence transition vectors. E: Enzymatic identification

Fig. 6 Targeted knockout of AtCLA1 gene by Csy4-FokI and FokI-Csy4 gene editing system M: 2 K PlusII DNA standard molecular weight. A: Results of CLCrV-A virus assay after successful transfer of Csy4-FokI editing vector into Arabidopsis thaliana. B: Results of CLCrV-A virus assays after successful transfer of the FokI-Csy4 editing vector into Arabidopsis thaliana. C: High-throughput detection of AtCLAI editing by the CLCrV-mediated Csy4-FokI system. D: Mutation types obtained after high-throughput sequencing analysis of the transiently transformed Csy4-FokI genome editing system HI-TOM

Fig. 7 Schematic diagram of MCP-FokI（FokI-MCP）and Csy4-FokI（FokI-Csy4）fusion protein A: MCP-FokI and FokI-MCP fusion proteins, FokI are located at the N and C ends of MCP respectively; B: Csy4-FokI and FokI-Csy4 fusion protein, FokI is located at the N terminal and C terminal of Csy4 respectively

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