sgRNA and cas9 protein were complexed and coinjected into one-cell stage zebrafish embryos. Approximately 1 nL of injection mix (10.5 μM sgRNA, 500 ng/μL cas9, 50 ng/μL donor plasmid and 50 μM NU7441 in 1% DMSO) was injected directly into each embryo. Embryos were then placed on ice-cold 1.5% agarose for no more than 15 min. NU7441 and cold exposure after injection were used to improve knock-in efficiency of mScarlet sequence by homology-directed repair mechanisms (HDR) [35]. NU7441 in 1% DMSO (50 μM) injected at the levels described did not have deleterious effects on normal zebrafish embryos, as assessed by morbidity, mortality and behavioral outcomes at 5 days post-fertilization (dpf) [36]. Injected embryos were collected, staged, and incubated in standard embryo media ((EM), 15 mM NaCl, 0.5 mM KCl, 1 mM MgSO4, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, 1 mM CaCl2 and NaHCO3 in fish system water).

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