Aggregate nuclease activity for LbCas12a RNP complexed with the three different crRNAs across ten different target sites with or without heatshock. Heatshock increases the median indel rates for all three crRNA frameworks. The highest median levels of nuclease activity are observed with the DRf-GC@13-crRNA. Deep sequencing data are from three independent zebrafish embryo injections performed by three different individuals. Each Box plot is drawn by GraphPad Prism, where the box represents the 25th and 75th percentile and the middle line is the median. Statistical significance is determined by one-way analysis of variance (ANOVA), “*” denotes P <0.05, “ns” denotes no significance (Supplementary Table 7). (B) Phenotypes obtained after the injection of the LbCas12a-crRNA RNP complexes targeting slc45a2 (albino) showing a strong albino phenotype (lower panel) compared to the WT uninjected animals (upper panel).

FnoCas12a–DRf-GC@13-crRNA RNP complexes achieve robust genome editing in zebrafish. (A) FnoCas12a–DRf-GC@13- crRNA RNP displays variable efficiency on ten different endogenous targets when injected at 24 fmol with or without heatshock. Error bars indicate ±s.e.m. (B) 34ÆC heatshock significantly increases FnoCas12a–DRf-GC@13-crRNA editing activity when the aggregate editing data are assessed across the ten target sites. For each dot plot the three lines represent 75th, 50th and 25th percentile, respectively. Deep sequencing data are from zebrafish embryos from three independent injections on different days by three different individuals (Supplementary Table 5). Statistical significance is determined by one-way analysis of variance (ANOVA), “****” denote P <0.0001 (Supplementary Table 7). (C) Phenotypes obtained after the injection of the FnoCas12a- DRf-GC@13- crRNA RNP complexes targeting slc45a2 (albino) showing a strong albino phenotype (lower panel) compared to the WT uninjected sibling animals (upper panel).