Figure 1

Figure 1. ErCas12a mediates efficient MMEJ-based knockin at the zebrafish tyr locus. (A) Schematic diagram of reporter gene knockin mediated by the MMEJ pathway through ErCas12a or Cas9 targeting at the tyr locus. The sequences of the overlapping target sites are displayed, with PAM sequences indicated in blue characters. (B) Indel efficiency of tyr ErCas12a site in embryos injected with ErCas12a mRNA and tyr pre-crRNA following 1 h heatshock at different developmental stages. (C) Comparison of indel efficiency of tyr Cas9 site with ErCas12a site with or without 1 h 34 °C heatshock at 3–4 hpf. HS: heatshock. Data in (B,C) represent mean ± s.d. of at least three independent replicates. Unpaired two-tailed Student’s t-test was used to calculate p values (* p < 0.05; ** p < 0.01; *** p < 0.001; “n.s.” indicates the difference is not significant). (D) Representative fluorescence expression of tyr knockin F₀ embryos obtained by injection of either ErCas12a or Cas9 MMEJ 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. Scale bar: 200 μm. (E) Evaluation of MMEJ-based knockin efficiency by the proportion of broad (Class I) and sparse (Class II) fluorescence-positive embryos injected with ErCas12a or Cas9 MMEJ-based knockin system at the tyr locus. 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 < 0.01; **** p < 0.0001).