DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

摘要

基于CRISPR/Cas9的DNA靶向很快成为合成生物学和基因组工程领域的一个领先工具。它利用由一个由RNA分子引导的细菌核酸内切酶“Cas9”能够定位几乎任何相匹配目标DNA序列的特性来进行结合和/解理。本期Nature上发表的一项研究，报告了利用单分子和宏观生化实验来揭示RNA-引导的Cas9定位DNA基因组（它们可以有数十亿碱基对长）内独特的20-碱基对序列的机制。该研究的结果突显了一个三核苷酸protospacer adjacent motif(简称PAM，在封面图片上为黄色）在将Cas9-RNA复合物向潜在DNA目标点上招募中以及在催化激活核酸酶（在封面图片上为棕色）中所起作用。目标DNA序列是通过一个“zip-up”机制被识别的，在此过程中RNA-DNA碱基对（红色）的序列信息抵消展开DNA双螺旋（紫色和蓝色）的能量成本。除了对于基因操纵的意义外，这项研究也揭示了DNA是怎样被Cas9-RNA(以细菌适应性免疫的一个效应子的角色）审查的。%The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA hetero-duplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.