Tsui Tsz Kin Martin, Hand Travis H, Duboy Emily C, Li Hong
Institute of Molecular Biophysics, ‡Department of Chemistry and Biochemistry, Florida State University , Tallahassee, Florida 32306, United States.
ACS Synth Biol. 2017 Jun 16;6(6):1103-1113. doi: 10.1021/acssynbio.7b00050. Epub 2017 Mar 20.
Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends on a 5'-NNNCC-3' PAM and is more efficient in cleaving negative supercoils than relaxed DNA. Kinetic as well as in vivo activity assays reveal that AceCas9 achieves optimal activity when combined with a guide RNA containing a 24-nucleotide complementarity region. The cytosine-specific, DNA topology-sensitive, and extended guide-dependent properties of AceCas9 may be explored for specific genome editing applications.
Cas9是一种正在积极开发用于基因组编辑和基因调控的RNA引导的DNA切割酶。双链DNA（即原间隔序列）要被Cas9切割，必须与Cas9结合的引导RNA的引导区域互补，该引导区域通常长度为20个核苷酸，并且与一个称为原间隔序列相邻基序（PAM）的短的Cas9特异性元件相邻。了解原间隔序列和PAM与Cas9相互作用的正确并列关系，将有助于开发通用且安全的基于Cas9的技术。我们报告了从嗜热栖热放线菌（AceCas9）中鉴定出Cas9并对其进行了生化特性分析。AceCas9依赖于5'-NNNCC-3'的PAM，并且在切割负超螺旋时比松弛DNA更有效。动力学以及体内活性测定表明，当AceCas9与包含24个核苷酸互补区域的引导RNA结合时，可实现最佳活性。AceCas9的胞嘧啶特异性、DNA拓扑敏感性和扩展的引导依赖性特性可用于特定的基因组编辑应用。
