FIGURE SUMMARY
Title

Chemical reprogramming enhances homology-directed genome editing in zebrafish embryos

Authors
Aksoy, Y.A., Nguyen, D.T., Chow, S., Chung, R.S., Guillemin, G.J., Cole, N.J., Hesselson, D.
Source
Full text @ Commun Biol

Overview of the in vivo homology-directed repair (HDR) detection system. a Schematic representation of the visual HDR readout. b The single guide RNA (sgRNA)-Cas9 complex targets the identical sequence in eGFP and eBFP2. Embryos are co-injected with HDR donor template to integrate the tdTomato transgene into the acta1-eBFP2 locus. c Confocal sections showing smyhc1:eGFP and acta1-eBFP2 expression and d merged images. Scale bars: 75 µm. e Cross-sectional representation of a zebrafish embryo showing slow and fast muscles. f Expression of smyhc1-eGFP in slow-muscle fibers (3 dpf). g Mosaic loss of smyhc1-eGFP expression in slow-muscle fibers (3 dpf) of embryos injected with a CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 complex targeting eGFP. Scale bars: 500 µm, main image and 100 µm, inset

Chemical enhancement of CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated homology-directed repair (HDR) efficiency. Effects of a SCR7, b NU7441, c RS-1 on the efficiency of CRISPR/Cas9-mediated tdTomato expression in zebrafish embryos. d Co-injection of optimized concentrations of NU7441 and RS-1 in zebrafish embryos. Error bars represent standard error of the mean. eg Fluorescent microscopy of zebrafish embryos injected with e tdTomato donor template, f CRISPR/Cas9 with tdTomato donor template, or g CRISPR/Cas9 with tdTomato donor template and optimized concentration of NU7441 at 50 µM. Scale bars: 250 µm

Single fiber analysis of homology-directed repair (HDR) events. Cross-sectional in vivo imaging of a zebrafish embryo (3 dpf) co-injected with NU7441 at 50 µM and RS-1 at 30 µM. a Merged image showing mutually exclusive expression of eBFP2 and tdTomato in individual fast-muscle fibers (yellow arrows). c, e Individual channels showing eBFP2 expression (c) and tdTomato (e). Adjacent slow-muscle fibers express eGFP. b, d, f Higher magnification confocal sections of regions indicated in a, c, e. Scale bars: 50 µm

To test if any random donor (tdTomato) insertion is seen when the donor is co-injected with a known Crispr (targeting golden locus) that is not specific to the eBFP2 target site, zebrafish embryos were co-injected with the following material: 1) (A) WT: Tomato 2) (B) WT: Golden Crispr/Ca9 + Tomato 3) (C) WT: Golden Crispr/Ca9 + Tomato + Drugs Multi-channel analysis of bright-field and red-fluorescence showed no red fibers in any of the experimental groups. Scale bars: 750 µm, main image and 250 µm, inset.

Acknowledgments
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