Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection
Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection
复制标题
DOI：
10.1016/j.celrep.2015.01.067
复制
发表时间：
2015-03-10
期刊：
影响因子：
8.8
通讯作者：
Seidel, Ralf
中科院分区：
文献类型：
--
作者：
Rutkauskas, Marius;Sinkunas, Tomas;Seidel, Ralf
CRISPR-Cas系统为细菌和古细菌提供了针对外源核酸的适应性免疫。在I型CRISPR-Cas系统中，入侵的DNA被称为Cascade的大型核糖核蛋白监视复合物检测到。Cascade的crRNA组分用于通过由碱基配对互补性驱动的R环的形成来识别外源DNA中的靶位点（原型间隔区）。使用单分子超螺旋实验与近碱基对分辨率，我们在这里探测R-环形成的机制，并检测短寿命的R-环中间体的脱靶位点轴承单错配。我们发现，R-环传播方向从原间隔区相邻的基序（PAM）。一旦达到不匹配，R环传播就会以长度相关的方式停止和崩溃。这明确地证明了R环的定向拉链通过快速拒绝与具有PAM近端突变的脱靶位点的结合来实现有效的靶识别。到达原型间隔区末端的R环被锁定以允许辅助Cas 3核酸酶/解旋酶降解DNA，而无需进一步的靶验证。
CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign nucleic acids. In type I CRISPR-Cas systems, invading DNA is detected by a large ribonucleoprotein surveillance complex called Cascade. The crRNA component of Cascade is used to recognize target sites in foreign DNA (protospacers) by formation of an R-loop driven by base-pairing complementarity. Using single-molecule supercoiling experiments with near base-pair resolution, we probe here the mechanism of R-loop formation and detect short-lived R-loop intermediates on off-target sites bearing single mismatches. We show that R-loops propagate directionally starting from the protospacer-adjacent motif (PAM). Upon reaching a mismatch, R-loop propagation stalls and collapses in a length-dependent manner. This unambiguously demonstrates that directional zipping of the R-loop accomplishes efficient target recognition by rapidly rejecting binding to off-target sites with PAM-proximal mutations. R-loops that reach the protospacer end become locked to license DNA degradation by the auxiliary Cas3 nuclease/helicase without further target verification.