Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Room 216, Main Building, No. 4, Section 2, North Jianshe Road, Chengdu, 610054, People's Republic of China.
Jiangsu Key Laboratory of Crop Genetics and Physiology, Co-Innovation Center for Modern Production Technology of Grain Crops, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou, 225009, China.
Genome Biol. 2018 Jul 4;19(1):84. doi: 10.1186/s13059-018-1458-5.
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.
在功能基因组学、精准医学和植物育种中，靶向特异性一直是应用基因组编辑系统的障碍。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。导致许多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过基于 WGS 的 Cpf1（Cas12a）脱靶分析。
我们对 T0 和 T1 代中由 Cas9 编辑的 34 株植物和由 Cpf1 编辑的 15 株植物以及 20 株不同的对照植物进行了 WGS 分析，在水稻中。测序深度范围从 45×到 105×，读映射率高于 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引起的，该过程导致每个植物约有 102 到 148 个单核苷酸变异（SNV）和约 32 到 83 个插入/缺失（indel）。在 12 个 Cas9 单指导 RNA（sgRNA）和 3 个 Cpf1 CRISPR RNA（crRNA）中，只有一个 Cas9 sgRNA 在 T0 系中在计算机程序预测的位点上导致脱靶突变。此外，由于 Cas9 或 Cpf1 与向导 RNA 在 T1 代中的持续表达，我们无法找到真正的脱靶突变的证据。
我们对多个样本类型的 WGS 数据进行了全面而严格的分析，表明 Cas9 和 Cpf1 核酸酶在产生靶向 DNA 修饰方面非常特异，并且通过设计高特异性的向导 RNA 可以避免脱靶。
