Abstract
CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.
Original language | English |
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Pages (from-to) | 416-424.e5 |
Journal | Molecular Cell |
Volume | 79 |
Issue number | 3 |
DOIs | |
State | Published - 6 Aug 2020 |
Keywords
- CRISPR-cas
- Candidate Phyla Radiation (CPR) bacteria
- Cas12c (C2c3)
- Cas12d (CasY)
- RuvC nuclease domain
- crRNA
- scoutRNA
- tracrRNA
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology