Cas 核酸酶识别特异性 PAM 的要求是基因组编辑的一个主要限制。SpCas9变异体 SpG 和 SpRY 分别识别 NGN 和 NRN PAM，在细胞培养和植物中有助于增加可编辑基因组位点的数量。然而，它们的应用还没有在动物身上得到证实。对斑马鱼和秀丽隐杆线虫的 SpG 和 SpRY 活性进行了表征和优化。SpG 和 SpRY 以 mRNA-gRNA 或核糖核蛋白(RNP)复合物的形式传递，能够在体内诱导突变，尽管在相同配方中的突变率低于 SpCas9。这种低活性是通过优化 mRNA-gRNA 或 RNP 浓度来克服的，从而在 SpCas9不能到达的区域进行有效的突变。我们还发现，CRISPRscan 算法可以预测 SpG 和 SpRY 活性。最后，我们应用 SpG 和 SpRY 技术，通过同源修复的方法产生了外源基因的敲入。总之，我们的研究结果扩展了针对动物基因组景观的 CRISPR-Cas。
图4.进一步的SpG和SpRY体内优化
The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAM, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. We have characterized and optimized the activity of SpG and SpRY in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to efficient mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm can predict SpG and SpRY activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.
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