Figure 3

Figure 3. ErCas12a mediates efficient NHEJ-based knockin at the zebrafish tbx2b locus. (A) Schematic diagram of R295H mutation and reporter gene knockin mediated by the NHEJ pathway through ErCas12a or Cas9 targeting at the tbx2b locus. Target site sequences are displayed, with PAM sequences indicated in blue. (B) Comparison of indel efficiency of tbx2b Cas9 site with ErCas12a site with or without 1 h heatshock at 34 °C. HS: heatshock. Data represent mean ± s.d. of at least three independent replicates. Unpaired two-tailed Student’s t-test was used to calculate p values (*** p < 0.001; “n.s.” indicates the difference is not significant). (C) Representative fluorescence expression of tbx2b knockin F₀ embryos obtained by injection of either ErCas12a or Cas9 NHEJ systems. Embryos with red fluorescence were separated into broad (Class I) and sparse (Class II) groups according to the expression pattern. Embryos with no fluorescence were designated Class III. The embryos are in lateral view with anterior to the left. Arrows indicate the regions showing red fluorescent signal. Scale bar: 200 μm. (D) Evaluation of NHEJ-based knockin efficiency by the proportion of broad (Class I) and sparse (Class II) fluorescence-positive embryos injected with ErCas12a or Cas9 systems at the tbx2b locus. The ErCas12a system injected embryos were separated into heatshocked (ErCas12a HS) and control (ErCas12a) groups. Number of embryos evaluated (n) is shown for each condition. Chi-square test was used to calculate p values between heatshocked (ErCas12a HS) group and others (**** p < 0.0001).