生物技术通报 ›› 2023, Vol. 39 ›› Issue (10): 68-79.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0248
邓嘉辉1(
), 雷建峰2, 赵燚1, 刘敏1, 胡子曜1, 尤扬子1, 邵武奎1, 柳建飞1, 刘晓东1()
收稿日期:2023-03-21
出版日期:2023-10-26
发布日期:2023-11-28
通讯作者:
刘晓东，男，博士，教授，研究方向：作物逆境分子生物学；E-mail: xiaodongliu75@aliyun.com作者简介:邓嘉辉，男，硕士，研究方向：作物逆境分子生物学；E-mail: 15754842489@126.com
基金资助:
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
Published:2023-10-26
Online:2023-11-28
摘要：
MCP是MS2噬菌体的外壳蛋白，Csy4是一种参与CRISPR 1-F系统crRNA生成的小型蛋白，能够以较高的特异性识别并结合RNA。目前CRISPR/Cas等基因组编辑技术存在靶向核酸酶分子量大、脱靶率高、受PAM位点限制等问题，为解决上述问题，构建基于上述两种小型蛋白的新型迷你基因组编辑系统。本研究采用AlphaFold2预测MCP-FokI、FokI-MCP、Csy4-FokI和FokI-Csy4融合蛋白的结构，通过浸花法将MCP-FokI和FokI-MCP编辑载体分别转化拟南芥，利用拟南芥叶片注射的方法投送CLCrV介导的Csy4-FokI与FokI-Csy4编辑系统，提取拟南芥基因组DNA，通过HI-TOM高通量测序检测新型迷你基因组编辑系统的编辑能力。结果显示，融合蛋白中MCP、FokI和Csy4都各自保持着自身原有的三维结构，预示它们都能正常发挥彼此的功能。构建靶向敲除拟南芥CLA1基因的4个不同中间间隔区的双靶位点MCP-FokI和MCP-FokI植物表达载体，初步证明MCP-FokI和FokI-MCP均不能实现对靶基因的靶向编辑。构建靶向敲除拟南芥CLA1基因的7个CLCrV介导的不同中间间隔区的双靶位点Csy4-FokI编辑载体，其中CLCrV介导的Csy4-FokI编辑系统能够实现对靶基因的靶向编辑，但是突变类型均为碱基置换类型且编辑效率很低，而FokI-Csy4基因组编辑体系并未检测到编辑的发生。成功构建了Csy4-FokI新型迷你基因组编辑系统，为克服CRISPR/Cas基因组编辑技术存在的问题提供了一种新的解决方案。
邓嘉辉, 雷建峰, 赵燚, 刘敏, 胡子曜, 尤扬子, 邵武奎, 柳建飞, 刘晓东. 基于Csy4与MCP的新型迷你基因组编辑系统的构建[J]. 生物技术通报, 2023, 39(10): 68-79.
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.
图1 MCP-FokI（FokI-MCP）和Csy4-FokI（FokI-Csy4）基因组编辑体系示意图 A：MS2-SL引导的FokI核酸酶原理概述：两个MCP-FokI融合蛋白招募两个不同的MS2-SL靶向到目标基因位点上，促进Fok I切割双链DNA断裂；B：sgRNA引导的FokI核酸酶原理概述：两个Csy4-FokI融合蛋白招募两个不同的sgRNA靶向到目标基因位点上，促进Fok I切割双链DNA断裂
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 |
表1 引物及用途
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 |
图2 融合蛋白三级结构预测 A：FokI-MCP与MCP-FokI融合蛋白结构预测图；B：FokI-Csy4与Csy4-FokI融合蛋白结构预测图
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
图3 MCP-FokI和FokI-MCP基因编辑载体构建图 M：2 K Plus II DNA标准分子量;WT：野生型；A：以拟南芥AtCLA1为靶标，对于同一个靶点分别用AtU6-26和AtU6启动子转录左侧guide-SL和右侧guide-SL；B：MCP-FokI和FokI-MCP编辑载体构建示意图；C：MCP-FokI和FokI-MCP编辑载体酶切鉴定结果；D：4个不同中间间隔区靶序列过渡载体酶切鉴定结果；E：酶切鉴定（1-8：AtU6-26∷SL1-35∷MCP-FokI-Ter-AtU6∷SL1-P1300, AtU6-26∷SL2-35∷MCP-FokI- Ter-AtU6∷SL2-P1300, AtU6-26∷SL3-35∷MCP-FokI-Ter-AtU6∷SL3-P1300, AtU6-26∷SL4-35∷MCP-FokI-Ter-AtU6∷SL4-P1300, AtU6-26∷SL1-35∷FokI- MCP-Ter-AtU6∷SL1- P1300, AtU6-26∷SL2-35∷FokI-MCP-Ter-AtU6∷SL2-P1300）
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
图4 MCP-FokI和FokI-MCP基因编辑系统靶向敲除AtCLA1 红色箭头表示转基因抗性苗
Fig. 4 Targeted knockout of AtCLA1 gene by MCP-FokI and FokI-MCP gene editing system The red arrow indicates transgenic resistant seedlings
图5 Csy4-FokI和FokI-Csy4基因编辑载体构建图 M：2K Plus II DNA标准分子量；WT：野生型；A：以拟南芥AtCLA1为靶标，对于同一个靶点分别用AtU6-26和AtU6启动子转录左侧sgRNA和右侧sgRNA；B：Csy4-FokI和Csy4-FokI编辑载体构建示意图；C：Csy4-FokI-Bzro和FokI-Csy4-B zro编辑载体酶切鉴定结果；D：7个不同中间间隔区靶序列过渡载体酶切鉴定结果；E：酶切鉴定（1-16：FokI-Csy4-CLCrV、Csy4-FokI-CLCrV, FokI-Csy4-sgRNA（1-5）-CLCrV、FokI-Csy4-sgRNA（2-5）-CLCrV, FokI-Csy4-sgRNA（2-6）-CLCrV, FokI-Csy4-sgRNA（3-5）-CLCrV, FokI-Csy4-sgRNA（3-6）-CLCrV, FokI-Csy4-sgRNA（4-5）-CLCrV, FokI-Csy4-sgRNA（4-6）- CLCrV, Csy4-FokI-sgRNA（1-5）-CLCrV, Csy4-FokI-sgRNA（2-6）-CLCrV, Csy4-FokI-sgRNA（2-6）-CLCrV, Csy4-FokI-sgRNA（3-5）-CLCrV, Csy4-FokI- sgRNA（3-6）-CLCrV, Csy4-FokI-sgRNA（4-5）-CLCrV, Csy4-FokI-sgRNA（4-6）-CLCrV）
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
图6 Csy4-FokI和FokI-Csy4基因编辑系统靶向敲除AtCLA1 M：2 K Plus II DNA 标准分子量；A：Csy4-FokI编辑载体成功转入拟南芥后CLCrV-A病毒检测结果（1-8：Csy4-FokI-CLCrV、Csy4-FokI-sgRNA（1-5）-CLCrV、Csy4-FokI-sgRNA（2-6）-CLCrV、Csy4-FokI-sgRNA（2-6）-CLCrV、Csy4-FokI-sgRNA（3-5）-CLCrV、Csy4-FokI-sgRNA（3-6）- CLCrV、Csy4-FokI-sgRNA（4-5）-CLCrV、Csy4-FokI-sgRNA（4-6）-CLCrV）；B：FokI-Csy4编辑载体成功转入拟南芥后CLCrV-A病毒检测结果（1-8：FokI-Csy4-CLCrV、FokI-Csy4sgRNA（1-5）-CLCrV、FokI-Csy4-sgRNA（2-6）-CLCrV、FokI-Csy4-sgRNA（2-6）-CLCrV、FokI-Csy4-sgRNA（3-5）-CLCrV、FokI-Csy4-sgRNA（3-6）-CLCrV、FokI-Csy4-sgRNA（4-5）-CLCrV、FokI-Csy4-sgRNA（4-6）-CLCrV）；C：CLCrV介导的Csy4-FokI体系对AtCLAI进行编辑的高通量检测；D：瞬时转化Csy4-FokI基因组编辑体系HI-TOM高通量测序分析后得到的突变类型
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
图7 MCP-FokI（FokI-MCP）和Csy4-FokI（FokI-Csy4）融合蛋白示意图 A：MCP-FokI和FokI-MCP融合蛋白，FokI分别位于MCP的N端和C端；B：Csy4-FokI和FokI-Csy4融合蛋白，FokI分别位于Csy4的N端和C端
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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