Fig. 2. Large-scale validation of the engineered CRISPR–Cas12f system.
a, Common sequences targetable by the SpCas9, AsCas12a and Cas12f systems and the gRNA formulations for each system. TTTR indicates TTTA or TTTG. b, A heatmap for indel frequencies per target obtained by SpCas9, AsCas12a or Cas12f. Measurement of indel frequencies in HEK293T cells transfected with SpCas9, AsCas12a, canonical Cas12f or engineered Cas12f vector constructs. Cells (1.75 × 105) were transfected with 2 μg of plasmid vector using a Fugene lipofection kit and grown for 96 h. c, A box-and-whisker plot for SpCas9-, AsCas12a- and Cas12f-induced indel frequencies merged with a dot plot. Whole data points (n = 88) were plotted with mean values as indicated by the horizontal cyan-colored line. Box plots represent the median with interquartile ranges (25–75%); whiskers extend to 1.5× the interquartile distance from the box. P values were derived by a Mann–Whitney U-test. NS, not significant. Error bars represent the s.d. d, Distribution of the number of targets per indel frequency subdivision. Values indicate the number of targets with indel efficiency belonging to the indicated ranges. The indel efficiencies and target information were provided in a source file and Supplementary Table 2. e, Distribution of the targets that show the highest efficiencies by the _ge3.0, _ge4.0 or _ge4.1 version. f, Schematic representation of the paired gRNA strategy for increasing Cas12f-mediated indel frequencies. DNA cleavages are centered in the spacing region, which is 10–30 bp in length. The size of the triangle indicates the frequency of a DNA-strand cleavage. g, Indel frequencies induced by Cas12f with engineered gRNA at ten targets when using either or both gRNAs. The upper and lower panels indicate indel frequencies for gRNAs with MS1/2 and MS1/2/3 engineering, respectively. h, Comparison of fold-changes in indel frequencies caused by MS1/2- and MS1/2/3-engineered gRNAs. The fold-changes were calculated from the indel frequencies induced by paired gRNAs compared with that of a gRNA that induces a higher indel frequency at a target located between the two paired gRNAs. i, Fold-changes in indel efficiencies by paired gRNAs according to the length of spacing.