Crop Bioengineering Center, Iowa State University, Ames, IA, USA.
Department of Agronomy, Iowa State University, Ames, IA, USA.
Plant Biotechnol J. 2019 Feb;17(2):362-372. doi: 10.1111/pbi.12982. Epub 2018 Jul 22.
CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9-guide RNA (gRNA) and LbCas12a-CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium-mediated transformation. On-target mutation analysis showed that 90%-100% of the Cas9-edited T0 plants carried indel mutations and 63%-77% of them were homozygous or biallelic mutants. In contrast, 0%-60% of Cas12a-edited T0 plants had on-target mutations. We then conducted CIRCLE-seq analysis to identify genome-wide potential off-target sites for Cas9. A total of 18 and 67 potential off-targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off-target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.
CRISPR/Cas9 和 Cas12a（Cpf1）核酸酶是植物中两种最强大的基因组编辑工具。在这项工作中，我们通过靶向玉米光亮 2 基因编码区来比较它们的活性，该区域具有两种核酸酶都能识别的重叠序列。我们使用农杆菌介导的转化将携带 SpCas9 向导 RNA（gRNA）和 LbCas12a-CRISPR RNA（crRNA）的构建体导入玉米自交系 B104 胚胎中。靶基因突变分析表明，90%-100%的 Cas9 编辑 T0 植物携带插入缺失突变，其中 63%-77%为纯合或双等位突变体。相比之下，0%-60%的 Cas12a 编辑 T0 植物有靶基因突变。然后，我们进行了 CIRCLE-seq 分析，以鉴定 Cas9 的全基因组潜在脱靶位点。两个 gRNA 分别鉴定出 18 和 67 个潜在的脱靶位点，与靶位点平均有 5 个错配。对选定的脱靶位点子集进行测序分析显示，在持续表达 Cas9 核酸酶和 gRNA 的 T1 植物中没有检测到突变。总之，我们的结果表明，本研究中使用的 CRISPR/Cas9 系统在玉米基因组编辑中具有高效特异性，而 CRISPR/Cas12a 需要进一步优化以提高编辑效率。
