中国水稻科学 ›› 2016, Vol. 30 ›› Issue (6): 577-586.DOI: 10.16819/j.1001-7216.2016.6024
杜彦修, 季新, 陈会杰, 彭廷, 张静, 李俊周, 孙红正, 赵全志*()
收稿日期:
2016-02-19
修回日期:
2016-06-03
出版日期:
2016-11-10
发布日期:
2016-11-10
通讯作者:
赵全志
基金资助:
Yan-xiu DU, Xin JI, Hui-jie CHEN, Ting PENG, Jing ZHANG, Jun-zhou LI, Hong-zheng SUN, Quan-zhi ZHAO*()
Received:
2016-02-19
Revised:
2016-06-03
Online:
2016-11-10
Published:
2016-11-10
Contact:
Quan-zhi ZHAO
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。
中图分类号:
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.
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' |
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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