Fig. 1
From: CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA
Determination of the ssDNA cleavage activities of the complex of Cas12a/crRNA/target DNA. a Time-course experiment of ssDNA (target-DNMT1-3) cleavage with crRNA-DNMT-23nt. The 3′-end FAM-labelled ssDNA was cleaved at expected sites (left); however, cleavage of 5′-FAM-labelled ssDNA generated no expected products (20 nt) but short oligonucleotides (<6 nt). b With a target-specific crRNA of crRNA-DNMT-23nt, Cas12a cleaved the 3′-FAM-labelled target-DNMT1-3 (ssDNA) at expected sites but cleaved the 5′-FAM-labelled target-DNMT1-3 (ssDNA) to short oligonucleotides. Collateral single-stranded target-DNMT1-3 (either 5′- or 3′-FAM-labelled) was cleaved to short oligonucleotides upon the formation of the ternary complex of Cas12a/crRNA-T1-24nt/target-T1 DNA. (c) Cleavage of random collateral ssDNA by the ternary complex of Cas12a/crRNA/target DNA. Random short single-stranded oligonucleotides (25 nt) were labelled with FAM at either the 5′-end or the 3′-end, obtaining N25-5′FAM and N25-3′FAM, respectively. The labelled random oligonucleotides were cleaved by the complex of Cas12a/crRNA-DNMT-23nt/target-DNMT1-3 (ssDNA) in lanes 3 and 4 or by the complex of Cas12a/crRNA-T1-24nt/target-T1 (ssDNA) in lanes 5 and 6, respectively. Lanes 1 and 2 showed synthesized random oligonucleotides that were labelled at the 5′-end and 3′-end, respectively. d Illustration of both cis- and trans-cleavage by the Cas12a complex in a–c. e Cis-cleavage of ssDNA (target-T1-R-FAM) by FnCas12a and its mutants. Mutations in D917, E1006, D1255 and R1218, four residues that are associated with the DNase activity, were generated. All tested mutants showed either completely lost or largely decreased cis-cleavage activity on target ssDNA. f Trans-cleavage of the 3′-FAM-labelled collateral ssDNA (target-DNMT1-3-R) by the ternary complexes of FnCas12a or its mutants (D917A, E1006A, D1255A and R1218A in FnCas12a). The trans-cleavage activity on collateral ssDNA was completely lost in E1006A and decreased in other mutants. g The ternary complex of Cas12a (PDB: 5B43) with proposed collateral ssDNA. Red dots represented the proposed positions of collateral ssDNA. Molecular graphic images were prepared using CueMol (http://www.cuemol.org). DNA was coloured in red, RNA was coloured in black and the RuvC catalytic pocket was indicated by dashed yellow circles
