Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences University of Copenhagen, Copenhagen, Denmark.
Core Facility for Integrated Microscopy (CFIM), Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Nat Commun. 2021 Jul 22;12(1):4476. doi: 10.1038/s41467-021-24707-3.
CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.
CRISPR-Cas12j 是一类从噬菌体中发现的新型微小 RNA 引导内切酶家族。这些核糖核蛋白提供了一个紧凑的支架，汇集了基因组编辑工具的所有关键活性。我们通过确定 Cas12j3/R-环复合物在 DNA 切割后的冷冻电镜结构，首次提供了 Cas12j 家族的结构见解。该结构揭示了 PAM 识别、杂交组装和 DNA 切割的机制。crRNA-DNA 杂交体被引导至间隔区，该间隔区将杂交体分开，引导 T 链朝向催化位点。保守的 RuvC 插入物锚定在间隔区，并与杂交体中 crRNA 的磷酸骨架相互作用。组装长度超过 12nt 的杂交体通过 RuvC 插入物中的关键功能残基激活催化。我们的发现表明了 Cas12j 在激活后如何引发非特异性 ssDNA 降解。定点突变分析支持 DNA 切割机制，为重新设计用于基因组编辑的 CRISPR-Cas12j 核酸酶提供了新途径。
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