New York Genome Center, New York, NY, USA; Department of Biology, New York University, New York, NY, USA.
New York Genome Center, New York, NY, USA; Department of Biology, New York University, New York, NY, USA.
Cell Rep. 2020 Mar 3;30(9):2859-2868.e5. doi: 10.1016/j.celrep.2020.02.010.
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.
一种广泛使用的 CRISPR 酶 S. pyogenes Cas9 的主要限制是在靶位点对严格的 NGG 原间隔基序（PAM）的要求。对于需要精确 Cas9 定位的基因组编辑应用来说，这种限制可能是有限的。最近，已经开发出两种具有放宽 PAM 要求（NG）的 Cas9 变体（xCas9 和 Cas9-NG），但它们的活性仅在少数内源性位点进行了测量。在这里，我们设计了一种高通量 Cas9 池竞争筛选方法，以比较 Cas9 变体在数千个基因组基因座上进行基因敲除、转录激活和抑制的性能。我们表明，PAM 的灵活性是以降低 DNA 靶向和切割的实质性代价为代价的。在这些 PAM 灵活的变体中，我们发现 Cas9-NG 无论基因组工程模式或 PAM 如何，都优于 xCas9。最后，我们将 xCas9 突变与 Cas9-NG 的突变相结合，创造出比现有 PAM 灵活的 Cas9 变体更强的转录调节剂。
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