Fig. 2: Engineering Cas12f1 endonuclease to increase the interaction with target gene and sgRNA.

a Summary of Cas12f1 variants with different combinations of mutations in the DNA-binding pocket. b Comparison of indel efficiency among Cas12f1_v1, Cas12f1_mini2, Cas12f1_mini3.1, and Cas12f1_mini4 at five gene targets. c Indel frequencies of Cas12f1_v1, Cas12f1_mini3.1, Cas12f1_mini4, and Cas12f1_v2 at twelve targets. d Sequence logo of twelve target sites where Cas12f1_v2 showed higher indel frequency compared to Cas12f1_v1, Cas12f1_mini3.1, and Cas12f1_mini4. Logo was created by WebLogos³⁹. e Position of the 6th nucleotide in the sgRNA spacer. TS, target strand; NTS, non-target strand; magenta letters, PAM sequence; light green letters, target sequence; light blue letters, sgRNA spacer sequence. f Location of G181 and T184 in Cas12f1.1 (PDB: 7C7L). G181.1, T184.1, and 6th nucleotide of the sgRNA spacer are highlighted in light green. g Heat map of relative indel frequencies of the seven mutated Cas12f1 variants at four gene targets. The seven variants contain different mutations in the G181 or T184 amino acid that interacts with the 6th nucleotide. The type of the 6th nucleotide is indicated in parentheses next to the gene name. h Additional experimental data for relative indel efficiency of G181K, G181R, and T184R variants at eight gene targets. The type of the 6th nucleotide is indicated in parentheses next to the gene name. i Comparison of indel frequencies of G181K, G181R, and T184R variants at eight gene targets from h (n = 8). j Comparison of indel frequencies of Cas12f1_v3 ~ 6 in ten gene targets where Cas12f1_v1 exhibited less than 20% editing efficiency. k Location of D143 and T147 in Cas12f1.1. D143, T147, and PAM are highlighted in light green. l Location of K330 in Cas12f1.2. K330 and A⁻⁸⁶ in Stem 1 of the sgRNA are highlighted in light green. m, n Comparison of indel efficiencies between the control variant, which exhibited higher editing efficiency for each target from Fig. 2j, and Cas12f1_v7 at ten gene targets. Data in the bar graphs represent the mean ± s.d. of three independent biological replicates. In the box-and-whisker plot, boxes represent the interquartile ranges with the median as a line, and whiskers extend from the minimum to the maximum values. P-values were obtained using the two-tailed Student’s t-test. ns, not significant. Source data are provided as a Source Data file.