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• W4383373445 endingPage "1047" @default.
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• W4383373445 abstract "Clustered regularly interspaced short palindromic repeats (CRISPR) sequences and CRISPR-associated (Cas) genes comprise CIRSPR-Cas effector complexes, which have revolutionized gene editing with their ability to target specific genomic loci using CRISPR RNA (crRNA) complementarity. Recognition of double-stranded DNA targets proceeds via DNA unwinding and base pairing between crRNA and the DNA target strand, forming an R-loop structure. Full R-loop extension is a prerequisite for subsequent DNA cleavage. However, the recognition of unintended sequences with multiple mismatches has limited therapeutic applications and is still poorly understood on a mechanistic level. Here we set up ultrafast DNA unwinding experiments on the basis of plasmonic DNA origami nanorotors to study R-loop formation by the Cascade effector complex in real time, close to base-pair resolution. We resolve a weak global downhill bias of the forming R-loop, followed by a steep uphill bias for the final base pairs. We also show that the energy landscape is modulated by base flips and mismatches. These findings suggest that Cascade-mediated R-loop formation occurs on short timescales in submillisecond single base-pair steps, but on longer timescales in six base-pair intermediate steps, in agreement with the structural periodicity of the crRNA–DNA hybrid. Here, using plasmonic DNA origami nanorotors, the authors observe in real time the R-loop formation and locking by the clustered regularly interspaced short palindromic repeats–Cas surveillance complex Cascade, thus reconstructing the underlying energy landscape and dynamics of this process." @default.
• W4383373445 created "2023-07-07" @default.
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• W4383373445 date "2023-07-01" @default.
• W4383373445 modified "2023-09-28" @default.
• W4383373445 title "The energy landscape for R-loop formation by the CRISPR–Cas Cascade complex" @default.
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• W4383373445 doi "https://doi.org/10.1038/s41594-023-01019-2" @default.
• W4383373445 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/37415009" @default.
• W4383373445 hasPublicationYear "2023" @default.
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