Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (10): 68-79.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0248
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DENG Jia-hui1(), LEI Jian-feng2, ZHAO Yi1, LIU Min1, HU Zi-yao1, YOU Yang-zi1, SHAO Wu-kui1, LIU Jian-fei1, LIU Xiao-dong1()
Received:
2023-03-21
Online:
2023-10-26
Published:
2023-11-28
Contact:
LIU Xiao-dong
E-mail:15754842489@126.com;xiaodongliu75@aliyun.com
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.
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
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 | |
T-Csy4-FokI-F | GGTCGGTATCCACGGAGTCCCAGCAGCCATGGGAGATCATTATCTTGATATTAG | 构建Csy4-FokI Construction of Csy4-FokI |
T-Csy4-FokI-R | GACGATCCGCCGGAGGACCCTCCAGAGAACCAAGGAACAAAACCTC | |
T-sgRNA1-F | TAGAGTCGACATAGCGATTGGTCATTAAGAATCACAATCGTTCACTGCCGTATAGGC | 构建AtU6∷sgRNA1 Construction of AtU6∷sgRNA1 |
T-sgRNA1-R | CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG | |
T-sgRNA2-F | GAGTCGACATAGCGATTGAATCACAATCATATCAGAGGTTCACTGCCGTATAGGC | 构建AtU6∷sgRNA2 Construction of AtU6∷sgRNA2 |
T-sgRNA2-R | CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG | |
T-sgRNA3-F | CTAGAGTCGACATAGCGATTGAAGAATCACAATCATATCAGTTCACTGCCGTATAGGC | 构建AtU6∷sgRNA3 Construction of AtU6∷sgRNA3 |
T-sgRNA3-R | CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG | |
T-sgRNA4-F | GAGTCGACATAGCGATTGAGAATCACAATCATATCAGGTTCACTGCCGTATAGGC | 构建AtU6∷sgRNA4 Construction of AtU6∷sgRNA4 |
T-sgRNA4-R | CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG | |
T-sgRNA5-F | GTCGAAGTAGTGATTGGCTTATGAAGCCATGAACAGTTCACTGCCGTATAGGC | 构建AtU6-26∷sgRNA5 Construction of AtU6-26∷sgRNA5 |
T-sgRNA5-R | TTGTCGACGCCCGGGATCCGGATCTCGAGTAAGAGCTCG | |
T-sgRNA6-F | GTAGTGATTGGAAGCCATGAACAACGCCGGTTCACTGCCGTATAGGC | 构建AtU6-26∷sgRNA6 Construction of AtU6-26∷sgRNA6 |
T-sgRNA6-R | TTGTCGACGCCCGGGATCCGGATCTCGAGTAAGAGCTCG | |
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 |
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 | |
T-Csy4-FokI-F | GGTCGGTATCCACGGAGTCCCAGCAGCCATGGGAGATCATTATCTTGATATTAG | 构建Csy4-FokI Construction of Csy4-FokI |
T-Csy4-FokI-R | GACGATCCGCCGGAGGACCCTCCAGAGAACCAAGGAACAAAACCTC | |
T-sgRNA1-F | TAGAGTCGACATAGCGATTGGTCATTAAGAATCACAATCGTTCACTGCCGTATAGGC | 构建AtU6∷sgRNA1 Construction of AtU6∷sgRNA1 |
T-sgRNA1-R | CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG | |
T-sgRNA2-F | GAGTCGACATAGCGATTGAATCACAATCATATCAGAGGTTCACTGCCGTATAGGC | 构建AtU6∷sgRNA2 Construction of AtU6∷sgRNA2 |
T-sgRNA2-R | CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG | |
T-sgRNA3-F | CTAGAGTCGACATAGCGATTGAAGAATCACAATCATATCAGTTCACTGCCGTATAGGC | 构建AtU6∷sgRNA3 Construction of AtU6∷sgRNA3 |
T-sgRNA3-R | CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG | |
T-sgRNA4-F | GAGTCGACATAGCGATTGAGAATCACAATCATATCAGGTTCACTGCCGTATAGGC | 构建AtU6∷sgRNA4 Construction of AtU6∷sgRNA4 |
T-sgRNA4-R | CCTTTCTAGAGCCCGGGATCCGGATCTCGAGTAAGAGCTCG | |
T-sgRNA5-F | GTCGAAGTAGTGATTGGCTTATGAAGCCATGAACAGTTCACTGCCGTATAGGC | 构建AtU6-26∷sgRNA5 Construction of AtU6-26∷sgRNA5 |
T-sgRNA5-R | TTGTCGACGCCCGGGATCCGGATCTCGAGTAAGAGCTCG | |
T-sgRNA6-F | GTAGTGATTGGAAGCCATGAACAACGCCGGTTCACTGCCGTATAGGC | 构建AtU6-26∷sgRNA6 Construction of AtU6-26∷sgRNA6 |
T-sgRNA6-R | TTGTCGACGCCCGGGATCCGGATCTCGAGTAAGAGCTCG | |
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 |
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. 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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