Rational sgRNA Design for CRISPR/Cas9-mediated Gene Inactivation. Mammalia

Components of clustered regularly interspersed palindromic repeat (CRISPR) loci, which function as an adaptable immune system in many prokaryotes, have recently been repurposed for use in mammalian cells. The Cas9 protein can be easily programed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but as yet there are few known rules governing on-target efficacy of this system. We created a pool of sgRNAs, tiling across all possible target sites of a panel of endogenous genes. By quantitatively assessing the ability of these sgRNAs to produce null alleles of their target gene, we discovered sequence features that improved activity, including a further optimization of the proto-spacer adjacent motif (PAM) for Cas9 from Streptococcus pyogenes. These results establish a predictive model of sgRNA activity beyond the NGG PAM sequence to improve sgRNA design for gene editing and genetic screens.

成簇规律间隔短回文重复序列（CRISPR）位点的组成元件，在众多原核生物中作为适应性免疫系统发挥功能，近年来已被重新改造并应用于哺乳动物细胞。Cas9蛋白可通过单引导RNA（sgRNA）进行便捷编程，以产生位点特异性DNA双链断裂，但目前已知的调控该系统靶标编辑效率的规则仍十分有限。我们构建了覆盖一组内源基因全部潜在靶标位点的sgRNA文库，通过定量评估这些sgRNA生成靶基因无效等位基因的能力，发现了可提升编辑活性的序列特征，其中包括对化脓性链球菌来源的Cas9的原间隔序列邻近基序（PAM）的进一步优化。本研究建立了超越NGG型PAM序列的sgRNA活性预测模型，可为基因编辑与遗传筛选中的sgRNA设计提供优化参考。