Fu Becky X H, St Onge Robert P, Fire Andrew Z, Smith Justin D
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
Stanford Genome Technology Center, Palo Alto, CA 94304, USA Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.
Nucleic Acids Res. 2016 Jun 20;44(11):5365-77. doi: 10.1093/nar/gkw417. Epub 2016 May 19.
Cas9, a CRISPR-associated RNA-guided nuclease, has been rapidly adopted as a tool for biochemical and genetic manipulation of DNA. Although complexes between Cas9 and guide RNAs (gRNAs) offer remarkable specificity and versatility for genome manipulation, mis-targeted events occur. To extend the understanding of gRNA::target homology requirements, we compared mutational tolerance for a set of Cas9::gRNA complexes in vitro and in vivo (in Saccharomyces cerevisiae). A variety of gRNAs were tested with variant libraries based on four different targets (with varying GC content and sequence features). In each case, we challenged a mixture of matched and mismatched targets, evaluating cleavage activity on a wide variety of potential target sequences in parallel through high-throughput sequencing of the products retained after cleavage. These experiments evidenced notable and consistent differences between in vitro and S. cerevisiae (in vivo) Cas9 cleavage specificity profiles including (i) a greater tolerance for mismatches in vitro and (ii) a greater specificity increase in vivo with truncation of the gRNA homology regions.
Cas9是一种与CRISPR相关的RNA引导核酸酶，已被迅速用作DNA生化和基因操作的工具。尽管Cas9与向导RNA（gRNA）之间的复合物在基因组操作方面具有显著的特异性和通用性，但仍会发生靶向错误事件。为了更深入了解gRNA与靶标的同源性要求，我们在体外和体内（在酿酒酵母中）比较了一组Cas9::gRNA复合物的突变耐受性。基于四个不同靶标（具有不同的GC含量和序列特征），用变异文库测试了多种gRNA。在每种情况下，我们对匹配和错配靶标的混合物进行挑战，通过对切割后保留产物的高通量测序，并行评估多种潜在靶标序列上的切割活性。这些实验证明了体外和酿酒酵母（体内）Cas9切割特异性图谱之间存在显著且一致的差异，包括（i）体外对错配的耐受性更高，以及（ii）体内随着gRNA同源区域的截断，特异性增加幅度更大。
