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作物学报

作物学报 ›› 2019, Vol. 45 ›› Issue (10): 1522-1534.doi: 10.3724/SP.J.1006.2019.84130

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于优化sgRNA系统提高海岛棉CRISPR/Cas9基因组编辑功效的研究

李继洋1,2,胡燕1,姚瑞2,代培红1,*(),刘晓东1,*()   

  1. 1新疆农业大学农学院 / 新疆农业大学农业生物技术重点实验室, 新疆乌鲁木齐 830052
    2石河子大学生命科学院, 新疆石河子 832000
  • 收稿日期:2018-10-16 接受日期:2019-05-12 出版日期:2019-10-12 网络出版日期:2019-09-10
  • 通讯作者: 代培红,刘晓东
  • 基金资助:
    本研究由南京农业大学-新疆农业大学联合基金项目(KYYJ201603);新疆农业大学作物学重点学科发展基金项目资助

Enhancing CRISPR/Cas9 genomic editing efficiency based on optimization of sgRNA of Gossypium barbadense L.

LI Ji-Yang1,2,HU Yan1,YAO Rui2,DAI Pei-Hong1,*(),LIU Xiao-Dong1,*()   

  1. 1Agricultural College, Xinjiang Agricultural University / Key Laboratory of Agricultural Biotechnology, Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China
    2Life Sciences College, Shihezi University, Shihezi 832000, Xinjiang, China
  • Received:2018-10-16 Accepted:2019-05-12 Published:2019-10-12 Published online:2019-09-10
  • Contact: Pei-Hong DAI,Xiao-Dong LIU
  • Supported by:
    This study was supported by the Nanjing Agricultural University-Xinjiang Agricultural University Joint Fund Project(KYYJ201603);Xinjiang Agricultural University Crop Science Key Discipline Development Fund Project.

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摘要：

CRISPR/Cas9基因组编辑体系已经在多种作物中被建立, 其最大的优势在于能简单高效定向创制突变体。然而, CRISPR/Cas9基因组编辑体系在实际操作过程中经常会出现没有编辑的情况, 或者靶向编辑目的基因的同时也会引发不同程度的脱靶效应, 这对CRISPR/Cas9基因组编辑技术的运用带来不利影响。本研究基于前期在海岛棉体细胞中建立的CRISPR/Cas9基因组编辑体系, 通过对构建Cas9不同密码子优化方式、不同PAM位点个数和不同靶位点数量的编辑载体, 来分析比较编辑效率和脱靶效应的差异。结果表明, 2种Cas9密码子不同优化方式的编辑载体产生的编辑效率和脱靶效应无显著差异; 优化后的双PAM位点的编辑效率显著高于单PAM位点, 且脱靶效率显著较低; 大部分双靶序列编辑效率均高于单靶序列且脱靶效率较低。上述研究结果为今后优化CRISPR/Cas9介导的海岛棉基因组编辑体系奠定了重要的理论依据。

关键词: 棉花, CRISPR/Cas9, 编辑效率, 脱靶效率, sgRNA类型

Abstract:

The CRISPR/Cas9 genome editing system has been established in many crops. The advantages of its directional creation of mutants are increasingly favored by researchers. However, the CRISPR/Cas9 genome editing technology targets the editing of the target gene and also triggers off-target effects at different frequencies, which determine the reliability of the genome editing system. This study was based on the previous CRISPR/Cas9 genome editing system established in the island-cotton somatic cells. Edit the vector by constructing different codon optimization methods for Cas9, different numbers of PAM sites and different target sites, and the difference in editing efficiency and off-target effect were analyzed and compared. There was no significant difference in editing effects and off-target effects caused by Cas9-edited vectors with different optimal codons. The partially double-sgRNA had significantly higher editing efficiency and significantly lower off-target efficiency than the single sgRNA; the editing efficiency of the transformed No shift sgRNA target sequence was significantly higher than that of the Shift sgRNA and the former had a significant decrease in off-target efficiency relative to the latter. Therefore, using No shift sgRNA type target sequence can effectively improve the editing efficiency and significantly reduce the off-target efficiency, thus laying a theoretical foundation for optimizing the CRISPR/Cas9 mediated island cotton genome editing system and accurately and efficiently creating island cotton functional gene mutants in the future.

Key words: cotton, CRISPR/Cas9, editing efficiency, off-target efficiency, sgRNA type

表1

本研究使用的引物信息"

序列名称
Sequence name		 序列
Sequence (5'-3')		 PAM位点序列
PAM site sequence
Shift GbU6-4PERA1-sg2F GATTGTCTTTCGCAGAATGCATGA CGG
Shift GbU6-4PERA1-sg2R AAACTCATGCATTCTGCGAAAGAC
GbU6-4PERA1-sg2F GATTGTTCGCAGAATGCATGACGG TGG
GbU6-4PERA1-sg2R AAACCCGTCATGCATTCTGCGAAC
GbU6-5PGGB-sg1F AAGTGTGTCGAAGTACTGAAGCGG CGG
GbU6-5PGGB-sg1R AAACCCGCTTCAGTACTTCGACAC
GbU6-4PGGB-sg2F GATTGTCTTTCAGTCTTATGATGG TGG
GbU6-4PGGB-sg2R AAACCCATCATAAGACTGAAAGAC
Shift GbU6-5PGGB-sg1F AAGTGCTCTGTCGAAGTACTGAAG CGG
Shift GbU6-5PGGB-sg1R AAACCTTCAGTACTTCGACAGAGC
Shift GbU6-4PGGB-sg2F GATTGTGTTCTTTCAGTCTTATGA TGG
Shift GbU6-4PGGB-sg2R AAACTCATAAGACTGAAAGAACAC
Test GGB-sg1F AAGTGGAAAGAGAATGGCGAC
Test GGB-sg1R AGCTAATTCGCTATTGCAATCAATC
Test GGB-sgR ACCATGTGATTCTAAACCAGG
Test ERA1-sg1F TAATAGGTGGTCAGGTTATGC
Test ERA1-sg1R AGGAGACTTGCAACCTGATC
FJ-1F GTAAAACGACGGCCAG
FJ-1R CAGGAAACAGCTATGAC
FJ-2F TAAACTGAAGGCGGGAAACG
FJ-2R CGGTTCTGTCAGTTCCAAACG

图1

No shift sgRNA和Shift sgRNA靶序列特点及其作用模式 红框标识为CRISPR/Cas9作用的PAM位点。"

图2

海岛棉基因组编辑载体结构示意图 A: 含Cas9I编辑载体; B: 含Cas9II编辑载体。"

表2

脱靶序列预测信息"

脱靶序列名称
Name for sequence of off target		 序列
Sequence (5°-3°)		 预测脱靶率
Predict the off target rate
OT-GGB-sgRNA1-1 GGTCCAAGGACTGAAGCTGTGG 0.311
OT-GGB-sgRNA1-2 GGTTGAAGTAGTAAAGCGGAGG 0.177
OT-Shift GGB-sgRNA1-1 TCCAGTCGAAATACTGAAGAGG 0.408
OT-Shift GGB-sgRNA1-2 CTCTGTTGATGAACTGATGGGG 0.287

表3

脱靶序列检测引物信息"

序列名称
Sequence name		 序列
Sequence (5°-3°)
OTtest-G1-1F CCTTGAAAGTTGCTTCCGAC
OTtest-G1-1R GAAACTGCCTTTTTTGACACC
OTtest-G1-2F CGAAAAATGGTTAGTGGTGAC
OTtest-G1-2R GTGGATGTTACTGTAGCAATG
OTtest-SG1-1F AGAGGACCAAACACATTAACC
OTtest-SG1-1R TTTGGGGGAGAGCACTTTTG
OTtest-SG1-2F ATCACCTTGCTACTCTTTCTG
OTtest-SG1-2R GTTTAGTTGACCAAGCATCTC

图3

海岛棉编辑载体酶切检测结果 A: GbU6-5P-GGB-sgRNA1编辑载体酶切检测结果; M1为1 kb plus marker。B: 1和2分别为GbU6-5P::GGB-sgRNA1-GbU6- 4P::GGB-sgRNA2和GbU6-5P::GGB-sgRNA1-GbU6-4P::ERA1- sgRNA2编辑载体酶切检测结果; M2为2 kb plus II marker。"

图4

海岛棉原生质体制备及其转化结果 A: 海岛棉转化前原生质体镜检结果; B: 海岛棉转化GbU6-5P::GGB-sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I载体后原生质体镜检结果。标尺为40 μm。"

图5

海岛棉转化不同靶序列编辑效应检测结果 A: 1为转化GbU6-5P-GGB-sgRNA1-Cas9I载体酶切前直接PCR扩增结果, 2为转化GbU6-5P-GGB-sgRNA1-Cas9I载体酶切后PCR扩增结果, 3为转化GbU6-5P-sgRNA1-Cas9I载体酶切前直接PCR扩增结果, 4为转化GbU6-5P-sgRNA1-Cas9I载体酶切后PCR扩增结果。B: 1为转化GbU6-5P::GGB-sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I直接PCR扩增结果, 2为转化GbU6-5P-sgRNA1-GbU6-4P-sgRNA2- Cas9I直接PCR扩增结果。C: G1、E1为转化GbU6-5P::GGB-sgRNA1-GbU6-4P::ERA1-sgRNA2-Cas9I酶切前直接PCR扩增结果, G2、E2为转化GbU6-5P::GGB-sgRNA1-GbU6-4P::ERA1-sgRNA2-Cas9I酶切后PCR扩增结果, G3、E3为转化GbU6-5P-sgRNA1-GbU6-4P- sgRNA2-Cas9I酶切前直接PCR扩增结果, G4、E4为转化GbU6-5P-sgRNA1-GbU6-4P-sgRNA2-Cas9I酶切后PCR扩增结果。M1为1 kb plus marker, M2为2 kb plus II marker。"

图6

海岛棉转化不同类型靶序列碱基突变测序结果 A: 转化GbU6-5P-GGB-sgRNA1-Cas9I载体靶位点编辑效应测序检测结果; B: 转化GbU6-5P::GGB-sgRNA1-GbU6-4P::ERA1- sgRNA2-Cas9I载体GGB靶位点编辑效应测序检测结果; C: 转化GbU6-5P::GGB-sgRNA1-GbU6-4P::ERA1-sgRNA2-Cas9I载体ERA1靶位点编辑效应测序检测结果; D: 转化GbU6-5P::GGB-sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I载体GGB-sgRNA2靶位点编辑效应测序检测结果; E: 转化GbU6-5P::GGB-sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I载体GGB-sgRNA1靶位点编辑效应测序检测结果。红色框为PAM位点的位置。"

图7

海岛棉转化不同类型靶序列碱基突变部分测序峰图 A: 转化GbU6-5P-GGB-sgRNA1-Cas9I靶序列基因组碱基突变测序峰图; B: 转化GbU6-5P::GGB-sgRNA1-GbU6-4P::ERA1- sgRNA2-Cas9I靶序列基因组碱基突变测序峰图; C: 转化GbU6-5P::GGB-sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I靶序列基因组碱基突变测序峰图。"

表4

转化不同靶序列编辑效率统计结果"

转化载体名称
Conversion carrier name		 一个位点碱基突变个数
A base muta- tion number		 2个位点同时突变个数
Number of mutations for two sites at the same time		 突变率
Mutation rate
sgRNA1位点
sgRNA1 site		 sgRNA2位点
sgRNA2 site		 sgRNA1位点sgRNA1 site sgRNA2位点sgRNA2 site
GGB-sgRNA1 12/95 — — 0.126 —
Shift GGB-sgRNA1 10/95 — — 0.105 —
GGB-sgRNA1-ERA1-sgRNA2 — 15/70 15/70 0.214 0.214
Shift GGB-sgRNA1-ERA1-sgRNA2 — 11/70 10/70 0.157 0.143
GGB-sgRNA1-GGB-sgRNA2 — 9/57 7/57 0.157 0.123
Shift GGB-sgRNA1-GGB-sgRNA2 — 7/60 5/60 0.117 0.083

图8

转化海岛棉不同类型靶序列的原生质体Cas9及对应sgRNA半定量检测结果 1、2分别为转化GbU6-5P-GGB-sgRNA1-Cas9I和对照编辑载体检测GGB-sgRNA1, 3、4为GbU6-5P-GGB-sgRNA1-GbU6-4P- ERA1-sgRNA2-Cas9I编辑载体分别检测GGB-sgRNA1、ERA1- sgRNA2, 5、6为转化GbU6-5P::GGB-sgRNA1-GbU6-4P::GGB- sgRNA2-Cas9I分别检测GGB-sgRNA1、GGB-sgRNA2, 7为转化与GbU6-5P-sgRNA1-4P-sgRNA1Cas9I对照编辑载体检测GGB- sgRNA1。"

图9

海岛棉脱靶目标序列扩增检测 转化No shift sgRNA类型靶序列: 1、2分别为转化GbU6-5P-GGB-sgRNA1-Cas9I和对照编辑载体, 3、4为GbU6-5P- GGB-GbU6-4P-sgRNA1- ERA1-sgRNA2-Cas9I和对照编辑载体, 5、6为转化GbU6-5P::GGB-sgRNA1-GbU6-4P::GGB-sgRNA2 -Cas9I和对照编辑载体。转化Shift sgRNA类型靶序列: 1、2分别为转化Shift GbU6-5P-GGB-sgRNA1-Cas9I和对照编辑载体, 3、4为Shift GbU6-5P-GGB-GbU6-4P-sgRNA1-ERA1-sgRNA2- Cas9I和对照编辑载体, 5、6为转化Shift GbU6-5P::GGB- sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I和对照编辑载体。"

图10

海岛棉脱靶目标序列突变检测 A: OTtest-SG1-2引物检测; 1为转化Shift GbU6-5P-GGB- sgRNA1-Cas9I编辑载体, 2为Shift GbU6-5P-GGB-GbU6-4P- sgRNA1-ERA1-sgRNA2-Cas9I编辑载体, 3为转化Shift GbU6- 5P::GGB-sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I编辑载体。 B: OTtest-G1-2引物检测; 1为转化GbU6-5P-GGB-sgRNA1-Cas9I编辑载体, 2为GbU6-5P-GGB-GbU6-4P-sgRNA1-ERA1-sgRNA2- Cas9I编辑载体, 3为转化 GbU6-5P::GGB-sgRNA1-GbU6-4P:: GGB-sgRNA2-Cas9I编辑载体。C: OTtest-SG1-1引物检测; 1为转化Shift GbU6-5P-GGB-sgRNA1-Cas9I编辑载体, 2为Shift GbU6-5P-GGB-GbU6-4P-sgRNA1-ERA1-sgRNA2-Cas9I编辑载体, 3为转化Shift GbU6-5P::GGB-sgRNA1-GbU6-4P::GGB-sgRNA2- Cas9I编辑载体。"

图11

海岛棉脱靶目标序列测序峰图 A、B、C分别为OTtest-SG1-2、OTtest-G1-2、OTtest-SG1-1引物扩增转化下图对应载体得到的目标片段测序结果。红框标识为碱基突变位点。"

表5

转化不同靶序列脱靶效率统计结果"

转化载体名称
Conversion carrier name		 脱靶序列检测Detection of off-target sequences
OTtest-G1-1 OTtest-SG1-1 OTtest-SG1-2
GGB-sgRNA1 6/50 — —
Shift GGB-sgRNA1 — 0 18/50
GGB-sgRNA1-ERA1-sgRNA2 0 — —
Shift GGB-sgRNA1-ERA1-sgRNA2 — 0 6/50
GGB-sgRNA1-GGB-sgRNA2 0 — —
Shift GGB-sgRNA1-GGB-sgRNA2 — 0 6/50
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