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. 2021 Dec 24;50(2):1162–1173. doi: 10.1093/nar/gkab1227

Figure 1.

Figure 1.

Addition of scaffold RNA stimulates target DNA cleavage by AsCas12a programmed with 5′ truncated crRNAs. (A) Schematic representation of Cas12a-mediated pre-crRNA processing. A subtype V-A CRISPR-Cas locus organization is shown (22). Genes coding for proteins responsible for interference (Cas12a) and adaptation (Cas4, Cas1, Cas2) are labeled and shown by colored arrows. The CRISPR array consists of spacers (colored rectangles) separated by repeats (black rhombi). Processing of the CRISPR array transcript (pre-crRNA) is carried out at the repeats by the Cas12a nuclease. (B) Schematics of the in vitro target DNA cleavage assay. A fragment of DNA containing the target site complementary to the crRNA spacer is amplified with PCR primers (indicated by arrows). Cleavage by AsCas12a programmed with crRNA recognizing the target is indicated by a red arrow and results in the appearance of cleavage products of indicated lengths. (C) The crRNA used for target recognition and cleavage. The scaffold moiety (black font) adopts a pseudoknot structure (12); the spacer sequence is highlighted in blue-coloured font. The numbers in red indicate the 5′-terminal nucleotides of truncated variants of crRNA tested. In the +2 variant, 18 nucleotides from the 3′-end of the spacer were eliminated. (D) Results of in vitro target cleavage assay with AsCas12a programmed with truncated crRNAs (indicated above the gel). The reactions contained 10 nM of the target, 500 nM of recombinant AsCas12a, and 5 μM of indicated crRNAs (a 1:50:500 ratio). Reactions were incubated 30 min at 37°C and products were resolved by agarose gel electrophoresis. Only the uncut target and one of the cleavage products (2776 bp) are shown. In the rightmost gel reactions were supplemented with 5 μM of scaffold or spacer RNA, as indicated. The results were quantified using ImageLab software. Mean cleavage efficiencies from four independent experiments and standard deviations are shown. (E,F) Kinetics and concentration dependence of target cleavage by AsCas12a programmed with full-sized (E) or –7 (F) crRNAs. Reactions proceeded for 1, 5, 10 and 20 minutes and were carried out at the indicated target:AsCas12a:crRNA ratios. Gels at the bottom of each panel show the results of cleavage assays conducted in the presence of equimolar amounts of scaffold crRNA moiety. Mean cleavage efficiencies and standard deviations from three independent experiments are shown on the right. (G) Target DNA cleavage by AsCas12a programmed with 3 μM full-sized, split, or separate scaffold and spacer crRNA moieties at a 1:30:300 target:AsCas12a:RNA ratio. ‘Split’ reactions contained both the scaffold and spacer RNAs. Mean cleavage efficiencies and standard deviations from three independent experiments are shown on the right. (H) Cleavage by AsCas12a programmed with increasing concentrations (5, 10 and 15 μM) of spacer RNA, corresponding to 1:50:500, 1:50:1000 and 1:50:1500 DNA:AsCas12a:crRNA ratios. Mean cleavage efficiencies and standard deviations calculated from three independent experiments are shown.