Fig. 2

Temperature is a key factor modulating Cpf1 activity in vitro and in vivo. a Schema illustrating different temperature incubations after Cpf1–crRNA RNP complex injections targeting slc45a2 (alb) and tyr. b Phenotypic evaluation of AsCpf1–crRNA RNP complex (10 fmol) injections at different temperature incubations (T). Stacked barplots showing the percentage of tyr-like (white), severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ ²-test (****p < 0.0001). c A representative picture showing 48-hpf-old embryos obtained after AsCpf1–crRNA RNP complex injections targeting tyr at different temperature incubations. Scale bar, 1.25 mm. d Phenotypic evaluation of LbCpf1–crRNA RNP complex (10 fmol) injections at different temperature incubations (T). Stacked barplots showing the percentage of alb/tyr-like (white), mosaic mutants (gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ ²-test (****p < 0.0001). e A representative picture showing 48-hpf-old embryos obtained after LbCpf1–crRNA RNP complex injections targeting slc45a2 at different temperature incubations. Scale bar, 1.25 mm. f Schematic illustrating different incubation conditions (0, 4, 8, or 24 h at 34 °C, then at 28 °C) after AsCpf1–crRNA RNP complex (10 fmol) injections targeting tyr in zebrafish (top). Phenotypic evaluation of AsCpf1–crRNA RNP complex injections targeting tyr in the conditions described above (bottom). Stacked barplots showing the percentage of tyr-like (white), severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ ²-test (****p < 0.0001). g Schematic illustrating different incubation conditions: 8 h at 28 °C, 16 h at 34 °C, and then 24 h at 28 °C (34 °C 8–24 hpf) or 24 h at 28 °C, then 24 h at 34 °C (34 °C 24–48 hpf) after AsCpf1–crRNA RNP complex (10 fmol) injections targeting tyr in zebrafish (top). Phenotypic evaluation of AsCpf1–crRNA RNP complex injections targeting tyr in the conditions described above (bottom). Stacked barplots showing the percentage of severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ ²-test (****p < 0.0001)