Soczek Katarzyna M, Cofsky Joshua C, Tuck Owen T, Shi Honglue, Doudna Jennifer A
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA.
Innovative Genomics Institute; University of California, Berkeley, CA, USA.
Nucleic Acids Res. 2025 Jan 11;53(2). doi: 10.1093/nar/gkae1192.
RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.
RNA引导的核酸内切酶参与了从适应性免疫到位点特异性转座等一系列过程，并彻底改变了基因组编辑。CRISPR-Cas9、-Cas12及相关蛋白利用引导RNA通过尚未完全了解的机制识别基因组DNA内约20个核苷酸的靶位点。我们运用结构和生化方法评估了Cas12a蛋白-引导RNA复合物识别DNA的早期步骤。我们在此表明，Cas12a通过弯曲DNA引发瞬时核苷酸翻转来启动DNA靶标识别，从而使核碱基暴露以进行DNA-RNA杂交。对捕获的Cas12a-RNA-DNA监测复合物的冷冻电镜结构分析以及基于荧光的构象探测表明，Cas12a诱导的DNA螺旋不稳定使得能够发现并结合靶标。尽管这些酶家族有着不同的进化起源和不同的RNA-DNA杂交方向性，但这种初始DNA询问机制类似于CRISPR-Cas9。我们的研究结果支持了一种模型，即RNA介导的DNA干扰始于CRISPR-Cas蛋白的瞬时结合导致的局部螺旋扭曲。
