Fu Becky Xu Hua, Hansen Loren L, Artiles Karen L, Nonet Michael L, Fire Andrew Z
Department of Genetics, Stanford University, Stanford, CA 94305, USA
Department of Pathology, Stanford University, Stanford, CA 94305, USA.
Nucleic Acids Res. 2014 Dec 16;42(22):13778-87. doi: 10.1093/nar/gku1102. Epub 2014 Nov 15.
To study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9::guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleavage. Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (albeit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in 'seed' sequences matching the proximal 8 bp of the guide. A second target region requiring close homology was located at the other end of the guide::target duplex (positions 13-18 relative to the PAM). Sequences flanking the guide+PAM region had measurable (albeit modest) effects on cleavage. In addition, the first-base Guanine constraint commonly imposed by gRNA expression systems has little effect on overall cleavage efficiency. Taken together, these studies provide an in vitro understanding of the complexities of Cas9-gRNA interaction and cleavage beyond the general paradigm of site determination based on the 'seed' sequence and PAM.
为了研究化脓性链球菌Cas9活性的靶序列特异性、选择性和反应动力学，我们在体外使用纯化的Cas9::向导RNA复合物对随机变异靶标文库进行了检测。切割动力学是非线性的，最初有一阵快速的活性，随后是较慢的持续切割。与最近对Cas9序列特异性的其他分析一致，我们观察到对于在PAM序列或与向导近端8个碱基对匹配的“种子”序列中发生突变的靶标，切割受到相当大（尽管不完整）的损害。另一个需要紧密同源性的靶标区域位于向导::靶标双链体的另一端（相对于PAM的位置13 - 18）。向导+PAM区域两侧的序列对切割有可测量（尽管适度）的影响。此外，gRNA表达系统通常施加的第一个碱基鸟嘌呤限制对整体切割效率影响很小。综上所述，这些研究提供了对Cas9-gRNA相互作用和切割复杂性的体外理解，超越了基于“种子”序列和PAM进行位点确定的一般模式。
