A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice.
A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice.
复制标题
大规模全基因组测序分析揭示了水稻中 Cas9 和 Cpf1 (Cas12a) 核酸酶的高度特异性基因组编辑
DOI：
10.1186/s13059-018-1458-5
复制
发表时间：
2018-07-04
期刊：
影响因子：
12.3
通讯作者：
Zhang Y
中科院分区：
文献类型：
--
作者：
Tang X;Liu G;Zhou J;Ren Q;You Q;Tian L;Xin X;Zhong Z;Liu B;Zheng X;Zhang D;Malzahn A;Gong Z;Qi Y;Zhang T;Zhang Y
靶向特异性一直是基因组编辑系统在功能基因组学、精准医学和植物育种中应用的障碍。在植物中，只有有限的研究使用全基因组测序（WGS）来测试Cas9的脱靶效应。许多已发现的突变的原因仍然存在争议。此外，迄今为止，尚未在任何高等生物中报道Cpf 1（Cas 12 a）的基于WGS的脱靶分析。我们在T0和T1代中对34株由Cas9编辑的植物和15株由Cpf 1编辑的植物沿着20株不同的对照植物进行了水稻WGS分析。测序深度范围为45×至105×，读段映射率高于96%。我们的结果清楚地表明，编辑植物中的大多数突变是由组织培养过程产生的，这导致每株植物约102至148个单核苷酸变异（SNV）和约32至83个插入/缺失（indel）。在WGS评估的12种Cas9单向导RNA（sgRNA）和3种Cpf 1 CRISPR RNA（crRNA）中，只有1种Cas9 sgRNA在T0系中在计算机程序预测的位点处导致脱靶突变。此外，我们无法找到真正的脱靶突变的证据，这是由于在T1代中Cas9或Cpf 1与向导RNA的持续表达。我们对多种样品类型的WGS数据进行了全面而严格的分析，表明Cas9和Cpf 1核酸酶在产生靶向DNA修饰方面都具有非常特异性，并且可以通过设计具有高特异性的指导RNA来避免脱靶。本文的在线版本（10.1186/s13059-018-1458-5）包含补充材料，可供授权用户使用。
Targeting specificity has been a barrier to applying genome editing systems in functional genomics, precise medicine and plant breeding. In plants, only limited studies have used whole-genome sequencing (WGS) to test off-target effects of Cas9. The cause of numerous discovered mutations is still controversial. Furthermore, WGS-based off-target analysis of Cpf1 (Cas12a) has not been reported in any higher organism to date. We conduct a WGS analysis of 34 plants edited by Cas9 and 15 plants edited by Cpf1 in T0 and T1 generations along with 20 diverse control plants in rice. The sequencing depths range from 45× to 105× with read mapping rates above 96%. Our results clearly show that most mutations in edited plants are created by the tissue culture process, which causes approximately 102 to 148 single nucleotide variations (SNVs) and approximately 32 to 83 insertions/deletions (indels) per plant. Among 12 Cas9 single guide RNAs (sgRNAs) and three Cpf1 CRISPR RNAs (crRNAs) assessed by WGS, only one Cas9 sgRNA resulted in off-target mutations in T0 lines at sites predicted by computer programs. Moreover, we cannot find evidence for bona fide off-target mutations due to continued expression of Cas9 or Cpf1 with guide RNAs in T1 generation. Our comprehensive and rigorous analysis of WGS data across multiple sample types suggests both Cas9 and Cpf1 nucleases are very specific in generating targeted DNA modifications and off-targeting can be avoided by designing guide RNAs with high specificity. The online version of this article (10.1186/s13059-018-1458-5) contains supplementary material, which is available to authorized users.
登录
查看更多内容
影响因子：
4.6
作者：
Endo A;Masafumi M;Kaya H;Toki S
通讯作者：
Toki S
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
影响因子：
46.9
作者：
通讯作者：
--