Whole genome sequencing reveals rare off-target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9-edited cotton plants.
Whole genome sequencing reveals rare off-target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9-edited cotton plants.
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DOI：
10.1111/pbi.13020
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发表时间：
2019-05
影响因子：
13.8
通讯作者：
Zhang X
中科院分区：
文献类型：
--
作者：
Li J;Manghwar H;Sun L;Wang P;Wang G;Sheng H;Zhang J;Liu H;Qin L;Rui H;Li B;Lindsey K;Daniell H;Jin S;Zhang X
CRISPR/Cas9系统已广泛应用于作物改良。然而，我们对Cas9特异性的理解在Cas9编辑的植物中非常有限。为了鉴定编辑的作物中的靶上和脱靶突变，我们描述了靶向三个基因的14株Cas9编辑的棉花植物以及三株阴性（Ne）对照和三株野生型（WT）植物的全基因组测序（WGS）。与WT、阴性和棉花参考基因组序列相比，在14株Cas9编辑的植物中总共检测到4188-6404个独特的单核苷酸多态性（SNP）和312-745个插入/缺失（indel）。由于这些变异中的大多数缺乏原型间隔区邻近基序（PAM），我们证明了Cas9编辑后的大多数变异是由于体细胞克隆变异或/和来自母本植物的预先存在/固有变异，而不是脱靶效应。在总共4413个潜在的脱靶位点中（允许20 bp sgRNA和3 bp PAM序列内≤5个错配），WGS数据显示只有4个是真正的脱靶indel突变，通过桑格测序验证。此外，WT的固有遗传变异可以产生新的脱靶位点并破坏PAM，这表明应该非常小心地设计sgRNA以使脱靶效应最小化。这些发现表明CRISPR/Cas9系统对棉花植物具有高度特异性。
The CRISPR/Cas9 system has been extensively applied for crop improvement. However, our understanding of Cas9 specificity is very limited in Cas9‐edited plants. To identify on‐ and off‐target mutation in an edited crop, we described whole genome sequencing (WGS) of 14 Cas9‐edited cotton plants targeted to three genes, and three negative (Ne) control and three wild‐type (WT) plants. In total, 4188–6404 unique single‐nucleotide polymorphisms (SNPs) and 312–745 insertions/deletions (indels) were detected in 14 Cas9‐edited plants compared to WT, negative and cotton reference genome sequences. Since the majority of these variations lack a protospacer‐adjacent motif (PAM), we demonstrated that the most variations following Cas9‐edited are due either to somaclonal variation or/and pre‐existing/inherent variation from maternal plants, but not off‐target effects. Of a total of 4413 potential off‐target sites (allowing ≤5 mismatches within the 20‐bp sgRNA and 3‐bp PAM sequences), the WGS data revealed that only four are bona fide off‐target indel mutations, validated by Sanger sequencing. Moreover, inherent genetic variation of WT can generate novel off‐target sites and destroy PAMs, which suggested great care should be taken to design sgRNA for the minimizing of off‐target effect. These findings suggested that CRISPR/Cas9 system is highly specific for cotton plants.
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DOI：
10.1016/j.cub.2011.07.002
发表时间：
2011-08-23
期刊：
Current biology : CB
影响因子：
--
作者：
Jiang C;Mithani A;Gan X;Belfield EJ;Klingler JP;Zhu JK;Ragoussis J;Mott R;Harberd NP
通讯作者：
Harberd NP
影响因子：
14.8
作者：
Hu J;Meyers RM;Dong J;Panchakshari RA;Alt FW;Frock RL
通讯作者：
Frock RL
影响因子：
27.5
作者：
Ma, Xingliang;Zhang, Qunyu;Liu, Yao-Guang
通讯作者：
Liu, Yao-Guang
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
12.3
作者：
Lawrenson T;Shorinola O;Stacey N;Li C;Østergaard L;Patron N;Uauy C;Harwood W
通讯作者：
Harwood W