Fig. 3 | Nature Communications

Fig. 3

From: CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing

Fig. 3

Catalytic dead SpCas9 (SpdCas9) proximal targeting increases Cpf1 activity. a Schematic diagram of proxy-CRISPR approach. Temperature may control Cpf1 activity to access and/or unwind genomic DNA, impeding AsCpf1 to access the genomic target in vivo at 28 °C. When SpdCas9 binds in the proximity of the AsCpf1 target, it facilitates the availability of AsCpf1 to cleave the inaccessible target. b Diagram illustrating crRNA 1 (orange) and a proximal sgRNA (purple) targeting tyr exon 1 (Supplementary Fig. 10c and Supplementary Data 1) in zebrafish (top). AsCpf1–crRNA and/or SpdCas9-sgRNA RNP complexes were injected into one-cell-stage embryos and then incubated at 28 °C for 48 h (bottom). c Phenotypic evaluation of the experiment described in (b). 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). d Diagram illustrating crRNA 2 (orange) and sgRNA 2 (purple) targeting alb exon 1 (Supplementary Data 1, Supplementary Figs. 3f and 8e) in zebrafish (top). LbCpf1–crRNA and/or SpdCas9-sgRNA RNP complexes were injected into one-cell-stage embryos and then incubated at 28 °C for 48 h (bottom). e Phenotypic evaluation of the experiment described in (d). Stacked barplots showing the percentage of alb-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)

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