Peter Debye Institute for Soft Matter Physics, Universität Leipzig, 04103, Leipzig, Germany.
Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekis ave. 7, Vilnius, 10257, Lithuania.
Nat Commun. 2023 Jun 20;14(1):3654. doi: 10.1038/s41467-023-38790-1.
CRISPR-Cas effector complexes enable the defense against foreign nucleic acids and have recently been exploited as molecular tools for precise genome editing at a target locus. To bind and cleave their target, the CRISPR-Cas effectors have to interrogate the entire genome for the presence of a matching sequence. Here we dissect the target search and recognition process of the Type I CRISPR-Cas complex Cascade by simultaneously monitoring DNA binding and R-loop formation by the complex. We directly quantify the effect of DNA supercoiling on the target recognition probability and demonstrate that Cascade uses facilitated diffusion for its target search. We show that target search and target recognition are tightly linked and that DNA supercoiling and limited 1D diffusion need to be considered when understanding target recognition and target search by CRISPR-Cas enzymes and engineering more efficient and precise variants.
CRISPR-Cas 效应因子复合物能够抵御外来核酸，并最近被用作在靶标位点进行精确基因组编辑的分子工具。为了结合并切割它们的靶标，CRISPR-Cas 效应因子必须在整个基因组中检查是否存在匹配的序列。在这里，我们通过同时监测复合物的 DNA 结合和 R 环形成来剖析 I 型 CRISPR-Cas 复合物级联的靶标搜索和识别过程。我们直接量化了 DNA 超螺旋对靶标识别概率的影响，并证明级联使用易化扩散进行靶标搜索。我们表明，靶标搜索和靶标识别紧密相连，并且在理解 CRISPR-Cas 酶的靶标识别和靶标搜索以及工程更高效和精确的变体时，需要考虑 DNA 超螺旋和有限的 1D 扩散。
