Fig. 2: SpCas12f1 DNA cleavage and binding activity in vitro.
From: Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells
a Supercoiled plasmid DNA cleavage at different temperatures (left panel) and cleavage dependence on gRNA spacer lengths at 45 °C (right panel). b SpCas12f1 cleavage of supercoiled and linear DNA forms in vitro. Source data are provided as a Source data file. c Run-off sequencing of SpCas12f1 cleavage products (cutting is centered around 22–24 bp 3’ from the PAM). NTS and TS represent non-target and target strands, respectively. d Dependence of dsDNA-binding efficiency on temperature. Nuclease inactivated or dead (d) SpCas12f1 (D228A) RNP was mixed with dsDNA and preincubated for 30 min at the temperatures shown. DNA binding was monitored by EMSA (electrophoretic mobility shift assay) and quantified. e Molecular masses of complexes measured by mass photometry. Colored dashed lines indicate the observed molecular weights for the different components: red—gRNA, green—dCas12f1–gRNA binary complex, blue—dCas12f1–gRNA–DNA ternary complex. The theoretical masses for the given complex stoichiometries are shown in brackets. For all experiments, SpCas12f1 or dSpCas12f1 RNP complexes were assembled using a gRNA with an 18 nt long spacer.
