Department of Chemistry & Chemical Biology and Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
Nat Biotechnol. 2013 Sep;31(9):839-43. doi: 10.1038/nbt.2673. Epub 2013 Aug 11.
The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.
RNA 可编程的 Cas9 内切酶在与 20 个碱基对的指导 RNA 互补的双链 DNA 上切割。Cas9 系统已被用于修饰多种细胞和生物体的基因组，证明了其作为一种简便的基因组工程工具的潜力。我们使用体外选择和高通量测序来确定 8 种指导 RNA：Cas9 复合物切割 10(12)个潜在的脱靶 DNA 序列中的每一个的倾向。选择结果预测了人类基因组中的五个脱靶位点，当表达两种指导 RNA：Cas9 复合物之一时，在 HEK293T 细胞中证实这些位点发生了基因组切割。与以前的模型不同，我们的结果表明，指导 RNA：Cas9 的特异性延伸超过 7-12 个碱基的种子序列。我们的结果还表明，在体外和细胞中，活性和特异性之间存在权衡，因为较短、活性较低的指导 RNA 比较长、活性较高的指导 RNA 更具特异性。高浓度的指导 RNA：Cas9 复合物可以切割含有 PAM 附近或内部突变的脱靶位点，而当酶浓度有限时，这些突变不会被切割。
