| Autor: |
Mohsin M, Naqvi, Laura, Lee, Oscar E Torres, Montaguth, Fiona M, Diffin, Mark D, Szczelkun |
| Rok vydání: |
2021 |
| Předmět: |
|
| Zdroj: |
Nature chemical biology. 18(9) |
| ISSN: |
1552-4469 |
| Popis: |
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is widely used for genome editing and diagnostics, so it is important to understand how RNA-guided DNA recognition activates the cleavage of the target strand (TS) following non-target-strand (NTS) cleavage. Here we used single-molecule magnetic tweezers, gel-based assays and nanopore sequencing to explore DNA unwinding and cleavage. In addition to dynamic and heterogenous R-loop formation, we also directly observed transient double-stranded DNA unwinding downstream of the 20-bp heteroduplex and, following NTS cleavage, formation of a hyperstable 'clamped' Cas12a-DNA intermediate necessary for TS cleavage. Annealing of a 4-nucleotide 3' CRISPR RNA overhang to the unwound TS downstream of the heteroduplex inhibited clamping and slowed TS cleavage by ~16-fold. Alanine substitution of a conserved aromatic amino acid in the REC2 subdomain that normally caps the R-loop relieved this inhibition but favoured stabilisation of unwound states, suggesting that the REC2 subdomain regulates access of the 3' CRISPR RNA to downstream DNA. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink