Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition.
Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition.
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DOI：
10.1016/j.molcel.2021.07.043
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发表时间：
2021-11-04
期刊：
影响因子：
16
通讯作者：
Chang L
中科院分区：
文献类型：
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作者：
Xiao R;Wang S;Han R;Li Z;Gabel C;Mukherjee IA;Chang L
V-K型CRISPR-Cas系统以Cas 12 k效应子为特征，具有天然失活的RuvC结构域，并与用于RNA引导的DNA转座的Tn 7样转座子相关联，是一种有前途的精确DNA插入工具。为了揭示靶DNA识别的机制，我们确定了来自蓝细菌Scytonema hofmanni的Cas 12 k与单向导RNA（sgRNA）和双链靶DNA复合的冷冻电镜结构。结合诱变和体外DNA转座试验，我们的结果揭示了识别GGTT PAM序列和Cas 12 k结构元件的机制，这些元件对RNA引导的DNA转座至关重要。这些结构和机制的见解应该有助于开发V-K型CRISPR转座子系统作为基因组编辑的工具。CRISPR相关转座酶（CAST）系统允许RNA引导的DNA插入基因组中的精确位置，并且是基因组编辑的有前途的工具。Xiao等人报道了Cas 12 k的结构，Cas 12 k是V-K CAST系统的CRISPR效应蛋白，并提出了对靶DNA识别和RNA引导转座的机制见解。
The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for precise DNA insertion. To reveal the mechanism underlying target DNA recognition, we determined a cryo-EM structure of Cas12k from cyanobacteria Scytonema hofmanni in complex with a single guide RNA (sgRNA) and a double-stranded target DNA. Coupled with mutagenesis and in vitro DNA transposition assay, our results revealed mechanisms for the recognition of the GGTT PAM sequence and the structural elements of Cas12k critical for RNA-guided DNA transposition. These structural and mechanistic insights should aid in the development of type V-K CRISPR-transposon systems as tools for genome editing. CRISPR-associated transposase (CAST) systems allow for RNA-guided DNA insertion at precise locations in genomes and are promising tools for genome editing. Xiao et al. report the structure of Cas12k, the CRISPR effector protein of the type V-K CAST system, and present mechanistic insights into target DNA recognition and RNA-guided transposition.
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