Waksman Institute of Microbiology, Rutgers, State University of New Jersey, Piscataway, NJ 08854;
Waksman Institute of Microbiology, Rutgers, State University of New Jersey, Piscataway, NJ 08854.
Proc Natl Acad Sci U S A. 2017 May 23;114(21):5443-5448. doi: 10.1073/pnas.1619926114. Epub 2017 May 8.
The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.
原核生物规律成簇间隔短回文重复序列（CRISPR）相关 9 （Cas9）内切酶可切割双链 DNA 序列，这些序列由向导 RNA 分子指定，并由原间隔序列邻近基序（PAM）侧翼，广泛用于各种生物体的基因组编辑。RNA 编程的 Cas9 通过扫描基因组 DNA 来定位靶位点。我们试图阐明在与向导 RNA 配对之前，靶 DNA 与向导 RNA 配对的初始 DNA 询问步骤的机制。使用荧光和生化测定法，我们研究了模拟该途径中早期中间产物的 Cas9/向导 RNA 与模型 DNA 底物的复合物，这些中间产物最终导致 Cas9/向导 RNA-DNA 复合物的形成。结果表明，Cas9/向导 RNA 与 PAM 的结合有利于分离几个 PAM 近端原间隔序列碱基对，从而允许向导 RNA 进行初始靶标询问。双链体的不稳定性部分由 Cas9/向导 RNA 对 PAM 附近未配对的非靶链 DNA 片段的亲和力介导。此外，我们的数据表明，从 PAM 到双螺旋 DNA 进入 Cas9/单指导 RNA（sgRNA）内部的短阈值距离以外的双链 DNA 的进入受到阻碍。我们认为，不利于双链 DNA 结合的相互作用会促进 Cas9/向导 RNA-DNA 复合物形成过程中 PAM 近端区域的 DNA 弯曲，从而进一步使原间隔体双链体不稳定。我们的分析得出的机制解释了 Cas9/sgRNA 复合物如何在有效定位正确靶序列的同时，还能有效地询问与正确 PAM 相关的许多非靶序列。
