biostudies-literatureHarrington LBHoward Hughes Medical InstituteNIAID NIH HHSNHGRI NIH HHSNational Institutes of HealthNational Science Foundation416-424.e5https://www.ebi.ac.uk/biostudies/studies/S-EPMC8196889biostudies-literatureUnknown79(3)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 esMolecular cellA scoutRNA Is Required for Some Type V CRISPR-Cas Systems.PMC8196889U01AI142817-02R01 AI092531U01 AI1428171817593RM1 HG009490Chen JSBanfield JFAl-Shayeb BDoudna JAWitte IPBurstein DHarrington LBPaez-Espino DKyrpides NCMa EGertz DfalseA scoutRNA Is Required for Some Type V CRISPR-Cas Systems.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 es2020-01-01T00:00:00Z2020 Aug2025-04-19T00:29:08.736Z2022-02-11T02:46:25.079ZS-EPMC81968893264536710.1016/j.molcel.2020.06.022