基于CRISPR/Cas9系统的OsbHLH116基因编辑及其脱靶效应分析

doi:10.16819/j.1001-7216.2016.6024

摘要/Abstract

摘要：

以水稻OsbHLH116 基因为编辑对象,根据基因编码区序列(CDS)在第一外显子区域设计长度为19 bp的sgRNA,化学合成sgRNA的寡核苷酸序列,然后与CRISPR/Cas9系统表达载体pBUN411连接,再用农杆菌介导法获得水稻转基因株系,最后利用酶切和测序相结合的方法对OsbHLH116 突变体进行了筛选鉴定和脱靶效应分析。结果表明,所构建的pBUN411-gRNA载体成功实现了对基因OsbHLH116 的定向编辑。酶切分析表明在选取的10株T₀代转基因苗中得到了6个OsbHLH116 突变单株。对6个突变单株进行了TA克隆测序分析,发现了纯合突变、双等位突变和杂合突变3种类型。酶切分析表明2个潜在脱靶位点均未发生脱靶效应。

关键词: 水稻, 基因编辑, CRISPR/Cas9, 脱靶效应, OsbHLH116

Abstract:

With OsbHLH116 as the editable object, the 19-bp sgRNA was designed in the exon 1 site according to the coding sequence (CDS) and the oligonucleotides of sgRNA were chemically synthesized and inserted into linearized plasmid pBUN411. The transgenic lines were obtained by Agrobacterium-mediated transformation method. Identification of mutants and off-target effects analysis of OsbHLH116 were conducted by combing restriction enzyme digestion with sequencing. The results showed that the recombinant vector of pBUN411-gRNA succeeded in oriented editing of OsbHLH116. The restriction enzyme analysis results indicated that we got 6 mutants from 10 randomly selected transgenic lines. Sequence analysis of the six mutants indicated that homozygous mutation, biallelic mutation and heterozygous mutation occured. After searching the rice genome using the target sequence with PAM, two highly identical sites were found. However, we did not observe any mutation at these sites by restriction enzyme.

Key words: rice, gene editing, CRISPR/Cas9, off-target effects, OsbHLH116

中图分类号:

Q755
S511.01

杜彦修, 季新, 陈会杰, 彭廷, 张静, 李俊周, 孙红正, 赵全志. 基于CRISPR/Cas9系统的OsbHLH116基因编辑及其脱靶效应分析[J]. 中国水稻科学, 2016, 30(6): 577-586.

Yan-xiu DU, Xin JI, Hui-jie CHEN, Ting PENG, Jing ZHANG, Jun-zhou LI, Hong-zheng SUN, Quan-zhi ZHAO. CRISPR/Cas9 System-based Editing of OsbHLH116 Gene and Its Off-target Effect Analysis[J]. Chinese Journal OF Rice Science, 2016, 30(6): 577-586.

图/表 9

表1 本研究所用引物序列

Table 1 Primer sequence used in the study.

引物名称 Primer name	序列 Sequence	用途 Usage
sgRNA-F	5'-GGCGCCTCCATCGGAGGAAGAGA-3'	靶序列合成Construction of target site
sgRNA-R	5'-AAACTCTCTTCCTCCGATGGAGG-3'
pBUN411-VF	5'-CCATGAAGCCTTTCAGGACATGTA-3'	载体构建验证Verification of vector construction
pBUN411-VR	5'-ACGCTGCAAACATGAGACGGAGAA-3'
Basta-F	5'-AAGCACGGTCAACTTCCGTA-3'	除草剂基因验证Verification of Bt
Basta-R	5'-GAAGTCCAGCTGCCAGAAAC-3'
OsbHLH116-F	5'-GTTGATGTGGCAAGGAGGAG-3'	靶点两侧序列扩增Amplification of target region
OsbHLH116-R	5'-TACGCACCAGACAGTTCACC-3'
LOC_Os01g01380-F	5'-ACAAGCAATGCAAATGTTGG-3'	脱靶位点扩增Off-target amplification
LOC_Os01g01380-R	5'-CTCTTCGCCCACACCATC-3'
Chr4:+30193341-F	5'-AATAGATCACGCCGTCAACC-3'	脱靶位点扩增Off-target amplification
Chr4:+30193341-R	5'-CGAGACGAATCTTTTGAGCA-3'

图1 pBUN411-gRNA表达载体LB和RB之间线性结构及gRNA靶序列合成 A-表达载体pBUN411-gRNA的LB和RB之间线性结构。竖线-酶切位点Bsa Ⅰ; RB-T-DNA右边界; OsU3 promoter-水稻U3启动子;gRNA-Sc-向导RNA支架; OsU3 terminator-水稻U3终止子; Ubi1 promoter-玉米泛素基因1(Ubi1)启动子; Cas9-Cas9基因; Nos terminator-农杆菌胭脂碱合成酶基因(Nos)终止子; 35S Promoter-花椰菜花叶病毒(CaMV)35S启动子; Bar-抗除草剂基因; LB-T-DNA左边界。B-gRNA靶序列互补双链DNA。方框内为gRNA靶点序列; 灰字体为EarⅠ酶切位点; 斜体为PAM序列(不在所构建载体上);小写字体为BsaⅠ酶切后黏性互补末端。

Fig. 1. Linear structure of pBUN411-gRNA expression vectors between LB and RB and the construction of target site. A, Physical map between RB and LB of pBUN411-gRNA expression vectors. Vertical lines stand for BsaⅠ restriction sites; RB, Right border of T-DNA; OsU3 promoter, Rice U3 promoters; gRNA-Sc, Guide RNA scaffold; OsU3 terminator, Rice U3 terminators; Ubi1 promoter, Maize ubiquitin gene promoter; Cas9, Cas9 gene; Nos Terminator, Agrobacterium tumefaciens nopaline synthase gene (Nos) terminator; 35S Promoter, Cauliflower mosaic virus (CaMV) 35S promoter; Bar, Herbicide resistance Bar gene; LB, Left border of T-DNA. B, The target sites of gRNA complementary with double-stranded DNA. The target sequences are in the box; Gray letters stand for EarⅠ restriction sites; Letters with italics stand for PAM (not in the expression vectors); The lowercase stand for sticky ends of BsaⅠ.

图2 pBUN411-gRNA表达载体菌落PCR 1~4-表达载体的4个菌落PCR; 5-pBUN411空载体(对照); M-DM2000 标记。

Fig. 2. Verification of pBUN411-gRNA expression vectors via colony PCR. 1-4, Four independent colonies to detect expression vectors; 5, pBUN411 empty vectors (negative contrast); M, DM2000 marker.

图3 10株T0代转基因植株PCR产物EarⅠ酶切分析结果 1-10—10株转基因植株; WT-野生型; M-DM1000 标记;-RE-未进行酶切;+RE-使用EarI酶切; 箭头示酶切后PCR片段。

Fig. 3. Mutation analysis of 10 T0 transgenic lines by EarⅠ digestion of PCR product. 1-10, 10 transgenic lines; WT, Wild type; M, DM1000 marker; -RE, PCR product without restriction enzyme digestion; +RE, EarⅠ digested PCR product; Arrowheads, PCR fragments after EarI digested.

图4 4号、7号和10号单株PCR产物测序峰图下划线-PAM序列TGG; 箭头-DNA预期被切割处; 方框—套峰。A-7号未突变单株PCR产物测序峰; B-10号双等位突变单株PCR产物测序峰; C-4号纯合突变单株PCR产物测序峰。

Fig.4. Sanger sequencing results for PCR products of number 4, 7 and 10. Underlines stand for PAM (TGG); The arrows stand for the intended cleavage site; The double peak phenomenon is in the box. A, Sanger sequencing results for PCR products of non-mutation number 7 ; B, Sanger sequencing results for PCR products of biallelic mutation number 10; C, Sanger sequencing results for PCR products of homozygous mutation number 4.

图5 野生型与6株突变体突变位点序列比对蓝色字体-gRNA靶序列; 黄色高亮-PAM序列; 红色删除线-缺失碱基; 红色小写字体-插入碱基; -表示缺失;+表示插入;(0/0)-PCR产物测序。

Fig. 5. DNA sequence alignment of wild type with six mutant versions. The gRNA target site is shown in blue; The yellow highlighting denote PAM; Red dashes deleted bases; Insertion nucleotides are shown in red lowercases; -, Deletion; +, Insertion; (0/0)-Sequencing by using PCR product.

图6 野生型与株突变体氨基酸序列比对蓝色直线和方框-碱性-螺旋-环-螺旋结构域; 红色高亮-氨基酸缺失; 黄色高亮-翻译终止。

Fig.6. Comparison of the amino acid sequences between wild type and OsbHLH116 mutants. Blue lines and boxes denote basic-helix-loop-helix domain; The red highlighting denote amino acid deletion; The yellow highlighting denote translation termination.

图7 推定的脱靶位点序列与靶位点序列比对分析方框内为PAM (NGG) 序列,红色字体为与靶序列比对不匹配碱基。

Fig. 7. Sequences comparative analysis between the putative off-target sites and target sites.

图8 10株T0代转基因植株脱靶位点酶切验证 1~10-10株转基因植株; WT-野生型; M-DM1000 标记; -RE-未进行酶切; +RE-使用EarI酶切; 箭头-酶切后PCR片段。A-基因LOC_Os01g01380脱靶位点酶切鉴定; B-Chr4:+30193341脱靶位点酶切鉴定。

Fig. 8. PCR/ restriction enzyme (PCR/RE) assay to detect off-target of 10 plants of T0 transgenic lines. 1-10, 10 transgenic lines; WT, Wild type; M, DM1000 Marker; -RE, PCR product without restriction enzyme digestion; +RE, EarI digested PCR product; Arrowheads, PCR fragments after EarI digestion. A, PCR/RE assay to detect off-target of LOC_Os01g01380; B, PCR/RE assay to detect off-target of Chr4: +30 193 341.

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