Department of Plant Pathology, Kansas State University, Manhattan, KS, USA.
Hard Winter Wheat Genetics Research Unit, USDA-ARS, Manhattan, KS, USA.
Plant Biotechnol J. 2021 Dec;19(12):2428-2441. doi: 10.1111/pbi.13669. Epub 2021 Jul 28.
The development of CRISPR-based editors recognizing distinct protospacer-adjacent motifs (PAMs), or having different spacer length/structure requirements broadens the range of possible genomic applications. We evaluated the natural and engineered variants of Cas12a (FnCas12a and LbCas12a) and Cas9 for their ability to induce mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a-induced mutations in the wheat genome, even though with a lower rate than that reported for SpCas9. The eight-fold improvement in the gene editing efficiency was achieved for LbCas12a by using the guides flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides and showed substantial increase after subjecting transgenic plants to high-temperature treatment. We successfully applied LbCas12a-MGE for generating heritable mutations in a gene controlling grain size and weight in wheat. We showed that the range of editable loci in the wheat genome could be further expanded by using the engineered variants of Cas12a (LbCas12a-RVR) and Cas9 (Cas9-NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9-NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9. In conclusion, our study reports a set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to modification using the original SpCas9 nuclease.
基于 CRISPR 的编辑器识别不同的原间隔邻近基序 (PAMs) 或具有不同间隔长度/结构要求的发展拓宽了可能的基因组应用范围。我们评估了 Cas12a（FnCas12a 和 LbCas12a）和 Cas9 的天然和工程变体，以评估它们在诱导控制小麦重要农艺性状的内源性基因突变方面的能力。与 FnCas12a 不同，LbCas12a 诱导小麦基因组中的突变，尽管突变率低于报道的 SpCas9。通过使用侧翼带有核酶的向导，并由柳枝稷的 RNA 聚合酶 II 启动子驱动，LbCas12a 的基因编辑效率提高了 8 倍。使用 LbCas12a 的多重基因组编辑 (MGE) 效率与使用单 RNA 向导获得的效率相似，并在对转基因植物进行高温处理后有了实质性的提高。我们成功地应用 LbCas12a-MGE 在小麦中控制粒大小和重量的基因中产生可遗传的突变。我们表明，通过使用分别识别 TATV 和 NG PAMs 的 Cas12a（LbCas12a-RVR）和 Cas9（Cas9-NG 和 xCas9）的工程变体，可以进一步扩展小麦基因组中可编辑的基因座范围，Cas9-NG 在针对具有非典型 PAMs 的靶标时显示出比 xCas9 更高的编辑效率。总之，我们的研究报告了一组经过验证的 Cas12a 和 Cas9 编辑器的天然和工程变体，用于靶向使用原始 SpCas9 核酸酶不易修饰的小麦基因组中的基因座。
