梨愈伤组织双靶点CRISPR/Cas9基因编辑系统的建立

doi:10.16420/j.issn.0513-353x.2020-1829

摘要/Abstract

摘要：

为了在梨愈伤组织中建立CRISPR/Cas9基因编辑体系,将proDAM3:GUS转入‘茄梨’愈伤组织,通过构建双靶点的CRISPR/Cas9基因编辑载体,利用农杆菌介导的遗传转化法侵染proDAM3:GUS转基因梨愈伤组织,通过测序及GUS染色的方法检验敲除效果,分析靶基因突变类型。结果表明,共有4个转基因株系在靶位点处发生了基因突变,编辑效率为66.7%。对所有基因编辑株系的有效克隆进行分析,结果显示sgRNA1在靶位点GUST1的突变频率达到71.4%,高于sgRNA2在靶位点GUST2突变频率的46.4%,说明sgRNA1可以更有效地与靶位点结合。测序结果表明,4个基因编辑株系中GUS基因均被敲除,基因突变的类型包含碱基缺失、插入及两个靶点之间大片段的缺失,不存在碱基替换。GUS染色结果显示,基因敲除后的GUS转基因愈伤完全呈白色,说明利用CRISPR/Cas9多靶点基因编辑系统可以在梨愈伤组织中实现高效的基因敲除。

关键词: 梨, 愈伤组织, 基因编辑, 多靶点, CRISPR/Cas9

Abstract:

This work aims to establish the CRISPR/Cas9 gene editing system using European pear calli,and provides a technical platform for functional genomics study and molecular breeding of pear. We edited theGUS gene in proDAM3:GUS transgenic pear calli using a dual-cut strategy. Based on the sequence characteristics of the GUS gene,two sgRNAs were designed to target the second exon of the GUS gene. The corresponding GUS-pYLCRISPR/Cas9 plasmids were constructed and transformed into the proDAM3:GUS transgenic calli by Agrobacterium-mediated transformation. The results from sequencing data revealed that four out of six independent transgenic lines carried mutations at the target sites,with mutation rates of 66.7%. A total of 56 successfully sequenced clones from the gene edited lines were generated,which indicated that sgRNA1 target had the higher mutation frequency(71.4%)than that of sgRNA2 target(46.4%). We further analyzed the mutation types of the four GUS editing lines,and found that #1,#2,#3 lines showed deletion and insertion of nucleotides,while #5 lines showed deletion of large fragments between two target sites,whereas no nucleotide substitution was observed in any edited line. To check the phenotype,GUS staining showed that the control calli were blue in color,while the four gene editing lines were white,indicating that the CRISPR/Cas9 system was a powerful and precise method to induce targeted mutagenesis in pear calli.

Key words: pear, calli, gene editing, multiple target sites, CRISPR/Cas9

中图分类号:

S661.2

杨锋, 杨钦淞, 高雨豪, 马云晶, 许英, 滕元文, 白松龄. 梨愈伤组织双靶点CRISPR/Cas9基因编辑系统的建立[J]. 园艺学报, 2021, 48(5): 873-882.

YANG Feng, YANG Qinsong, GAO Yuhao, MA Yunjing, XU Ying, TENG Yuanwen, BAI Songling. Establishment of Dual-cut CRISPR/Cas9 Gene Editing System in Pear Calli[J]. Acta Horticulturae Sinica, 2021, 48(5): 873-882.

图/表 7

图1 GUS基因中CRISPR/Cas9靶位点的位置示意图及序列信息蓝色方框为外显子,灰色方框为内含子,绿色和橙色方框分别代表两个靶位点,方框下方的数字为位置信息,不同颜色的方框宽度按照序列长度比例绘制。下方的序列为靶位点信息,方向均为5′-3′。红色字体代表PAM序列,括号中的+/-号表示核酸酶识别的DNA链。

Fig. 1 Illustration of the target sites and sequences of CRISPR/Cas9 in GUS gene The blue bars represent the exons,the gray bar represents the intron,the green and orange bars represent two target sites,the numbers below the bar are position information,and the width of the bars are drawn in proportion to the sequence length. The sequences below the bars are the sequences of the target sites,which are shown in the direction of 5′-3′. The red font represents the PAM sequences,and the“+/-”in parentheses represent the DNA strand recognized by the nuclease.

表1 本研究中所用的引物

Table 1 All primers used in this study

用途 Usage	引物名称 Primer name	引物序列(5′-3′) Sequence
靶点接头正反向引物 The sense and reverse primers for the target with the adapters	GUSAtU3dT1-F	GTCATTGAGGTCGAAGACGCCAC
	GUSAtU3dT1-R	AAACGTGGCGTCTTCGACCTCAA
	GUSAtU3bT2-F	GTCACGATAGCACCCTCCCGGTG
	GUSAtU3bT2-R	AAACCACCGGGAGGGTGCTATCG
第1轮PCR 1st PCR	U-F	CTCCGTTTTACCTGTGGAATCG
第1轮PCR 1st PCR	gR-R	CGGAGGAAAATTCCATCCAC
第2轮PCR 2nd PCR	Pps-R	TTCAGAGGTCTCTACCGACTAGTATGGAATCGGCAGCAAAGG
	Pgs-2	AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC
	Pps-2	TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG
	Pgs-L	AGCGTGGGTCTCGCTCGACGCGTATCCATCCACTCCAAGCTC
载体测序引物 The sequencing primers for the vector	SP-R	TGCAATAACTTCGTATAGGCT
载体测序引物 The sequencing primers for the vector	SP-L2	GTCGTGCTCCACATGTTGACCG
阳性转化体筛选引物 The screening primers for positive transformants	Cas9-F	CTGACGCTAACCTCGACAAG
阳性转化体筛选引物 The screening primers for positive transformants	Cas9-R	CCGATCTAGTAACATAGATGACACC
基因编辑鉴定引物 The primers for identifying the gene editing events	GUS-F	ATGGTAGATCTGAGGGTAAATTTCTAGT
	GUS-R	GTTCTTGTAGCCGAAATCTGGAATG

图2 GUS -pYLCRISPR/Cas9载体结构图 LB:左边界;RB:右边界;T35s:35S终止子;Tnos:nos终止子;HPT:潮霉素磷酸转移酶基因;35S:花椰菜花叶病毒 CaMV35S启动子;Cas9p:SpCas9基因;NLS:核定位信号;AtU3d、AtU3b:拟南芥U3d、U3b启动子;T1、T2:GUS突变靶位点序列。

Fig. 2 Schematic diagram of GUS -pYLCRISPR/Cas9 vector LB:Left border;RB:Right border;T35s:35S terminator;Tnos:nos terminator;HPT:hygromycin B phosphotransferase;35S:Cauliflower mosaic virus 35S promoter;Cas9p:SpCas9 gene;NLS:Nuclear localization signal;AtU3d,AtU3b:U3d,U3b promoter fromArabidopsis;T1,T2:GUS Target sequence.

图3 proDAM3:GUS转基因株系的DNA鉴定(A)与GUS染色鉴定(B)

Fig. 3 The PCR(A)and GUS staining(B)confirmations of the fragment insertion of the proDAM3:GUS transgenic line

表2 基因编辑株系的突变类型分析

Table 2 Indel types in GUS mutant lines

突变类型InDel types	碱基数/bp Number of base	GUST1 sites(%)	GUST2 sites(%)	对应株系Line#
碱基插入Insertion	1 ~ 2	12(30%)	2(7.7%)	#1、#2、#3
单碱基缺失Single base deletion	1	5(12.5%)	8(30.8%)	#1、#2、#3
小片段缺失Small fragment deletion	2 ~ 15	7(17.5%)	0	#1、#2
大片段缺失Long fragment deletion	625	16(40.0%)	16(61.5%)	#5

图4 基因编辑株系#1、#2、#3(A)和#5(B)靶位点的突变类型蓝色方框为外显子,灰色方框为内含子,绿色和橙色方框分别代表两个靶位点,方框下方的数字为位置信息,不同颜色的方框宽度按照序列长度比例绘制。A:黑字体为PAM序列;蓝色字体为靶位点序列;红色字体代表基因编辑突变位点;B:靶位点用加粗字体表示,删除部分和插入序列用红色字体表示。

Fig. 4 The mutation types of #1,#2,#3(A)and #5(B)gene editing lines The blue bars represent the exons,the gray bar represents the intron,the green and orange bars represent two target sites,the numbers below the bar are position information,and the width of the bars are drawn in proportion to the sequence length. A:PAM sequence is shown in bold font;the target sites are shown in blue color;the mutations are represented in red color;B:The target sites are indicated by bold font,and the deletion and insertion are indicated by red color.

图5 基因编辑株系#1,#2,#3和#5的GUS染色结果

Fig. 5 GUS staining of results of #1,#2,#3 and #5 gene editing lines

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