High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene Knockout and Transcriptional Modulation.
High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene Knockout and Transcriptional Modulation.
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DOI：
10.1016/j.celrep.2020.02.010
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发表时间：
2020-03-03
期刊：
影响因子：
8.8
通讯作者：
Sanjana NE
中科院分区：
文献类型：
--
作者：
Legut M;Daniloski Z;Xue X;McKenzie D;Guo X;Wessels HH;Sanjana NE
广泛使用的CRISPR酶S.化脓性链球菌Cas9是靶位点处NGG前间隔区邻近基序（PAM）的严格要求。该约束对于需要精确的Cas9定位的基因组编辑应用可能是限制性的。最近，已经开发了具有宽松PAM要求（NG）的两种Cas9变体（xCas 9和Cas9-NG），但是仅在少量内源性位点处测量了它们的活性。在这里，我们设计了一种高通量Cas9合并竞争筛选，以比较Cas9变体在数千个基因组基因座上的基因敲除、转录激活和抑制性能。我们表明，PAM的灵活性是以降低DNA靶向和切割为代价的。在PAM柔性变体中，我们发现Cas9-NG优于xCas 9，无论基因组工程模式或PAM如何。最后，我们将联合收割机xCas 9突变与Cas9-NG的突变相结合，产生了比现有PAM柔性Cas9变体更强的转录调节剂。Cas9在其DNA靶位点需要NGG前间区序列邻近基序（PAM）。在这里，Legut等人对Cas9和两种最近开发的PAM柔性变体进行了基准测试，表明PAM的灵活性降低了功效。作者还报告了一种混合酶，它结合了来自两种PAM柔性变体的突变，证明了它对转录激活的改善功效。
A key limitation of the widely used CRISPR enzyme S. pyogenes Cas9 is the strict requirement of an NGG protospacer-adjacent motif (PAM) at the target site. This constraint can be limiting for genome editing applications that require precise Cas9 positioning. Recently, two Cas9 variants with a relaxed PAM requirement (NG) have been developed (xCas9 and Cas9-NG), but their activity has been measured at only a small number of endogenous sites. Here, we devise a high-throughput Cas9 pooled competition screen to compare the performance of Cas9 variants at thousands of genomic loci for gene knockout, transcriptional activation, and inhibition. We show that PAM flexibility comes at a substantial cost of decreased DNA targeting and cleavage. Of the PAM-flexible variants, we find that Cas9-NG outperforms xCas9 regardless of genome engineering modality or PAM. Finally, we combine xCas9 mutations with those of Cas9-NG, creating a stronger transcriptional modulator than existing PAM-flexible Cas9 variants. Cas9 requires an NGG protospacer-adjacent motif (PAM) at its DNA target site. Here, Legut et al. benchmark Cas9 and two recently developed PAM-flexible variants, showing that PAM flexibility comes with reduced efficacy. The authors also report a hybrid enzyme combining mutations from both PAM-flexible variants, demonstrating its improved efficacy for transcriptional activation.
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