背景回顾：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. 

主要研究：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. 

结果1-天然：Unlike FnCas12a, LbCas12a induced mutations in the wheat genome, even though with a lower rate than that reported for SpCas9.

结果2-变体：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. 

结果3-成功案例：We successfully applied LbCas12a-MGE for generating heritable mutations in a gene controlling grain size and weight in wheat.

结果4-编辑范围拓展：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. 

结论：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.

 摘 要 

基于CRISPR的基因组编辑器能够识别不同的原型间隔区相邻基序PAM，或者具有不同间隔区长度/结构要求，拓宽了潜在的基因组应用范围。作者评估了天然的和人为修改后的Cas12a（FnCas12a和LbCas12a）和Cas9，在诱导小麦重要农艺性状相关基因上突变的能力。与FnCas12a不同，LbCas12a能够诱导小麦基因组的突变，尽管基因编辑效率低于SpCas9。作者进一步利用侧翼序列为核糖酶的向导和柳枝稷RNA聚合酶Ⅱ启动子进行驱动，将LbCas12a的基因编辑效率提升了8倍。并且，利用LbCas12a进行多重位点基因组编辑（MGE）的效率与利用单一向导RNA获得的效率基本相当，并在转基因植株经高温处理后表现出显著的提高。作者成功地在小麦中应用了LbCas12a-MGE，对控制小麦粒径和重量的基因产生了可遗传的突变。作者发现，利用Cas12a（LbCas12a-RVR）和Cas9（Cas9-NG和xCas9）变体，即分别识别的PAM序列为TATV和NG，可以进一步扩大小麦基因组中可编辑位点的范围，并且Cas9-NG在非典型PAMs靶点上的编辑效率高于xCas9。综上，本文的研究报道了一组经过验证的天然的和人为修改后的Cas12a和Cas9编辑器，可用于小麦基因组中不能使用原始SpCas9核酸酶进行编辑的位点。