Although the Cas9 protein from Streptococcus pyogenes (SpCas9) is the most widely used clustered regularly interspaced short palindromic repeats (CRISPR) variant in genome engineering experiments, it does have certain limitations. First, the stringent requirement for the protospacer adjacent motif (PAM) sequence limits the target DNA that can be manipulated using this method in insects. Second, its complementarity specifications are not very stringent, meaning that it can sometimes cause off‐target effects at the target site. A recent study reported that an evolved SpCas9 variant, xCas9(3.7), with preference for various 5′‐NG‐3′ PAM sequences not only has the broadest PAM compatibility but also has much greater DNA specificity and lower genome‐wide off‐target activity than SpCas9 in mammalian cells. Here we applied the CRISPR/xCas9 system to target the white gene in Drosophila melanogaster, testing the genome‐editing efficiency of xCas9 at different PAM sites. On the GGG PAM site, xCas9 showed less activity than SpCas9. For the non‐NGG PAM site TGA, xCas9 could produce DNA cleavage and indel‐mediated disruption on the target gene. However, for other non‐NGG PAM sites, xCas9 showed no activity. These findings show that the evolved Cas9 variant with broad PAM compatibility is functional in Drosophila to induce heritable gene alterations, increasing the targeting range for the applications of genome editing in insects.

中文翻译：

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在果蝇中通过CRISPR / xCas9系统进行靶向基因破坏。

尽管来自化脓链球菌的Cas9蛋白（SpCas9）是基因组工程实验中使用最广泛的簇状规则间隔的短回文重复序列（CRISPR），它确实具有一定的局限性。首先，对原间隔子相邻基序（PAM）序列的严格要求限制了可使用此方法在昆虫中操纵的目标DNA。其次，它的互补性规格不是很严格，这意味着它有时会在目标部位造成脱靶效应。最近的一项研究报告说，进化出的SpCas9变体xCas9（3.7）偏爱各种5'-NG-3'PAM序列，不仅具有最广泛的PAM兼容性，而且具有更大的DNA特异性和更低的全基因组脱靶在哺乳动物细胞中比SpCas9具有更高的活性。在这里，我们将CRISPR / xCas9系统应用于白色在果蝇中的基因，测试了xCas9在不同PAM位点的基因组编辑效率。在GGG PAM网站上，xCas9的活动少于SpCas9。对于非NGG PAM位点TGA，xCas9可能在目标基因上产生DNA切割和插入缺失介导的破坏。但是，对于其他非NGG PAM站点，xCas9没有显示活动。这些发现表明，具有广泛PAM兼容性的进化Cas9变体在果蝇中具有功能，可诱导可遗传的基因改变，从而增加了在昆虫中进行基因组编辑的靶向范围。