Fig. 3: Characterization of NovaIscB activity in mammalian cells.

a, Indel formation mediated by WT OrufIscB, OrufIscB-REC and NovaIscB using 20-nt guides at six target sites across the human genome. Data are presented as mean ± s.d.; n = 3 replicate transfections. b, Specificity analysis based on the average indel fold changes of OrufIscB-REC or NovaIscB relative to WT OrufIscB and average percentage of off-target reads per guide from TTISS using a pool of four 20-nt guides each with WT OrufIscB, OrufIscB-REC and NovaIscB. The numbers of detected off-target (OT) sites are included in brackets. Error bars denote s.d.; n = 4 target sites. c, Sequences of two detected on-target and related detected off-target sites from TTISS. The percentages of reads related to the on-target site and each off-target site out of the total reads corresponding to each guide were included. Mismatched nucleotides in each off-target site relative to each on-target site are shown. Off-targets found with NovaIscB are shown in black. Gray shading denotes the TAM region. n.d., not detected. d, Indel formation mediated by NovaIscB, two engineered versions of OgeuIscB (enOgeuIscB and eIscB), AsCas12f–YHAM and SpCas9 with guides targeting HPRT1, DYNC1H1, CA2 and ANGPTL3. For NovaIscB, AsCas12f and SpCas9, 20-nt guides were used, and for enOgeuIscB and eIscB, 16-nt guides were used. Data are presented as mean ± s.d.; n = 3 replicate transfections. e, Fraction of on-target reads measured by TTISS for NovaIscB, enOgeuIscB, eIscB, AsCas12f–YHAM and SpCas9. Each point represents a different guide, individually transfected for each system compared. Data are presented as mean ± s.d. among different target genes; n = 3 target sites (AsCas12f–YHAM); n = 4 target sites (NovaIscB, enOgeuIscB, eIscB and SpCas9). *P < 0.05; **P < 0.01; NS, P > 0.05 from two-sided t-test for NovaIscB versus others. The P values are 0.00990 (enOgeuIscB), 0.01914 (eIscB), 0.70565 (AsCas12f–YHAM) and 0.66636 (SpCas9).