水稻CRISPR/Cas12a系统的优化及其介导的腺嘌呤碱基编辑器的建立

doi:10.13560/j.cnki.biotech.bull.1985.2021-0231

生物技术通报 ›› 2021, Vol. 37 ›› Issue (6): 279-285.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0231

水稻CRISPR/Cas12a系统的优化及其介导的腺嘌呤碱基编辑器的建立

王敬文¹(), 严芳¹, 柳浪¹, 周雪平¹^,², 王道文³^,⁴, 周焕斌¹^,⁵()

1.中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京 100193
2.浙江大学农业与生物技术学院,杭州 310058
3.中国科学院遗传与发育生物学研究所植物细胞与染色体工程国家重点实验室,北京 100101
4.河南农业大学农学院,郑州 450002
5.农业农村部桂林作物有害生物科学观测实验站,桂林 541399

收稿日期:2020-03-01 出版日期:2021-06-26 发布日期:2021-07-08
作者简介:王敬文,硕士研究生,研究方向:基因组编缉技术开发与应用;E-mail: wangjingwenHN@163.com
基金资助:
国家自然科学基金项目(31871948);中国农业科学院科技创新工程(Y2020PT26)

Optimization of CRISPR/Cas12a System and Development of It-mediated Adenine Base Editor in Rice

WANG Jing-wen¹(), YAN Fang¹, LIU Lang¹, ZHOU Xue-ping¹^,², WANG Dao-wen³^,⁴, ZHOU Huan-bin¹^,⁵()

1. State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193
2. College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058
3. State Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101
4. College of Agronomy,Henan Agricultural University,Zhengzhou 450002
5. Scientific Observing and Experimental Station of Crop Pests in Guilin,Ministry of Agriculture and Rural Affairs,Guilin 541399

Received:2020-03-01 Published:2021-06-26 Online:2021-07-08

摘要/Abstract

摘要：

为了构建高效的CRISPR/Cas12a介导的水稻基因编辑系统,通过测试不同来源的Cas12a蛋白、crRNA长度和构型来探索影响Cas12a编辑活性的因素发现,相对于AsCas12a和LbCas12a在crRNA为23 nt时具有更高的编辑效率,并且crRNA 3'端的U₄AU₄结构并不能增强其在水稻中的编辑活性,同时利用大肠杆菌腺嘌呤脱氨酶变体TadA8e开发了CRISPR/LbCas12a介导的腺嘌呤碱基编辑系统pUbi∶rBE58,成功地将水稻内源基因OsCPK15实现A到G替换(A>G),其效率为33.33%。本研究证明了利用CRISPR/Cas12a可以对水稻基因组进行编辑,丰富了水稻基因编辑工具,拓宽了基因编辑工具箱。

关键词: CRISPR, Cas12a, 腺嘌呤碱基编辑器, 水稻

Abstract:

In order to develop an efficient CRISPR/Cas12a-mediated gene editing system in rice,different Cas12a homologous proteins and crRNA configurations and lengths were tested to explore the factors affecting the editing activity of Cas12a. We found that LbCas12a had a high editing efficiency with crRNA 23 nt compared with AsCas12a,and the U₄AU₄ sequence at the 3' end of crRNA didn’t enhanced its editing activity. Meanwhile,we fused the Escherichia coli adenine deaminase variant TadA8e to develop the CRISPR/LbCas12a-mediated adenine base editing system pUbi∶rBE58,which successfully induced the conversion A to G at the target loci in rice endogenous gene OsCPK15,with an efficiency of 33.33%. Our results demonstrate that using CRISPR/Cas12a-mediated may genetically edit the genes of rice,and this enriches rice gene editing tools and broadens gene editing toolkits.

Key words: CRISPR, Cas12a, adenine base editor, Oryza sativa

王敬文, 严芳, 柳浪, 周雪平, 王道文, 周焕斌. 水稻CRISPR/Cas12a系统的优化及其介导的腺嘌呤碱基编辑器的建立[J]. 生物技术通报, 2021, 37(6): 279-285.

WANG Jing-wen, YAN Fang, LIU Lang, ZHOU Xue-ping, WANG Dao-wen, ZHOU Huan-bin. Optimization of CRISPR/Cas12a System and Development of It-mediated Adenine Base Editor in Rice[J]. Biotechnology Bulletin, 2021, 37(6): 279-285.

图/表 6

表1 实验中涉及的质粒和菌株

Table 1 Plasmids and strains involved in this study

	Name	Source
Plasmid	pUC57:LbCas12a-crRNA	Bought from Sangon Biotech
	pUC57:AsCas12a-crRNA	Bought from Sangon Biotech
	pUC57:LbCas12a	Bought from Sangon Biotech
	pUC57:AsCas12a	Bought from Sangon Biotech
	pENTR4	Lab stock
	pHZ11	This study
	pHZ4	This study
	pUC19:rBE3	Lab stock
	pUC19:LbCas12a	This study
	pUC19:AsCas12a	This study
	pUbi:rBE3	Lab stock
	pUbi:LbCas12a	This study
	pUbi:AsCas12a	This study
	pUbi:rBE58	This study
Strain	JM109	Lab stock
	DB3.1	Lab stock
	EHA105	Lab stock

图1 载体示意图 A:Cas12蛋白表达元件示意图;B:crRNA载体示意图;C:腺嘌呤碱基编辑载体(pUbi:rBE58)示意图

Fig.1 Schematics of gene editing constructs A:Schematics of Cas12a expression components. B:Schematics of crRNA vector. C:Schematics of the adenine base editor(pUbi:rBE58)

图2 利用水稻原生质体检测CRISPR/AsCas12a和CRISPR/LbCas12a的核酸酶活性 A:CRISPR/AsCas12a体系介导水稻原生质体中对OsCERK1进行基因编辑的PCR/RE检测;B:CRISPR/LbCas12a体系介导水稻原生质体中对OsCERK1进行基因编辑的PCR/RE检测

Fig.2 Detection of CRISPR/LbCas12a in rice protoplasts and the nuclease activity of CRISPR/AsCas12a A:PCR/RE detection of editing OsCERK1 gene in the rice protoplasts mediated by CRISPR/AsCas12a system. B:PCR/RE detection of editing OsCERK1 gene in the rice protoplasts mediated by CRISPR/LbCas12a system

表2 实验中所用靶位点

Table 2 Target sites used in this study

Gene name	Target site sequence
OsCERK1	AGGAGGCCCTCAACTCGCCGGAT
OsCPK13	ATGGGCAACGCATGCGGCGGTTC
OsCPK17	ATAGGATCGTGCAGCGCGGGCAC
OsCPK15	CACCACTAGATATAGAAGGCCAT
Bsrd1	AATTTTAACTGATAAATATAAGT
OsJAZ4	AGGACGAGATCATGGAGTCTGAC

图3 不同crRNA构型对AsCas12a和LbCas12a核酸酶活性的比较

Fig.3 Comparison of nuclease activities of AsCas12a and LbCas12a with different crRNA configurations

图4 rBE58对水稻内源基因OsCPK15的靶位点腺嘌呤碱基编辑 A:靶位点OsCPK15 的各种编辑类型,靶序列、PAM序列、预期的靶碱基T和突变后的碱基分别用加粗、绿色、红色和蓝色字体标识,峰图中突变后的碱基用红色下划线标识;B:rBE58介导OsCPK15靶位点的碱基编辑活性框内靶碱基编辑效率汇总表

Fig.4 Targeted adenine base editing of endogenous gene OsCPK15 in rice using the rBE58 system A:Various editing types of target site OsCPK15 generated by rBE58,target sequences,PAM sequences,targeted base T in the putative editing window,and detected A-to-G conversions are highlighted in bold,green,red,and blue,respectively;and the detected mutated nucleotide in the figure are indicated by the red underlines. B:Summary of target base editing efficiency in the activity window of base editing for the target sites ofOsCPK15 gene mediated by rBE58

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水稻CRISPR/Cas12a系统的优化及其介导的腺嘌呤碱基编辑器的建立

Optimization of CRISPR/Cas12a System and Development of It-mediated Adenine Base Editor in Rice

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