Figure 5

Figure 5. T5exo-ErCas12a efficiently mediates heatshock-free indel mutation and large fragment knockin. (A) Schematic diagram of ErCas12a fused with T5 exonuclease at the N-terminus. The two proteins are separated by an Xten linker. (B) Indel efficiency in embryos injected with T5exo-ErCas12a mRNA and tyr pre-crRNA under heatshock or non-heatshock conditions. The results were compared with ErCas12a mRNA injection under the same conditions. (C) Indel efficiency in embryos injected with T5exo-ErCas12a mRNA and alb pre-crRNA2 under heatshock or non-heatshock conditions. The results were compared with ErCas12a mRNA injection under the same conditions. 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) Evaluation of MMEJ-based knockin efficiency by the proportion of broad (Class I) and sparse (Class II) fluorescence-positive embryos injected with T5exo-ErCas12a, Cas9, or ErCas12a (with and without heatshock) systems at the tyr locus. Representative images of different expression patterns are shown in Figure 1D. HS: Heatshock. (E) Evaluation of NHEJ-based knockin efficiency by the proportion of broad (Class I) and sparse (Class II) fluorescence-positive embryos injected with T5exo-ErCas12a, Cas9, or ErCas12a (with and without heatshock) systems at the tbx2b locus. Representative images of different expression patterns are shown in Figure 3C. HS: Heatshock. Number of embryos evaluated (n) is shown for each condition. Chi-square test was used to calculate p values between T5exo-ErCas12a group and others in (D,E) (* p < 0.05; **** p < 0.0001; “n.s.” indicates the difference is not significant).