Pigmentation loss after injection of Cas9 or Cpf1 RNPs into zebrafish embryos.

Left side, diagram of experiment. 1-cell zebrafish embryos were injected with 1 nL of either Cas9 or Cpf1 injection mix targeting the tyrosinase gene (tyr). Cas9 injection mix was 10 uM SpCas9 protein (NEB), 150 pg sgRNAs (n=3, made using NEB EnGen sgRNA kit), 50 mM KCl, 2.5 mM MgCl2, 5 mM Tris pH 8, 0.01% phenol red. Cpf1 injection mix was 10 uM LbCpf1 protein (NEB), 150 pg crRNAs (n=3, from Synthego), 50 mM KCl, 2.5 mM MgCl2, 5 mM Tris pH 8, 0.01% phenol red. Injected embryos were grown at 28C. Survival rate was scored at 24 hours-post-fertilization (hpf); both injections had &gt;85% survival. Larvae were anesthetized at 72 hpf and scored for pigmentation. <br>Right side, images of representative uninjected, Cas9 injected and Cpf1 injected larvae, with phenotype scoring indicated in the lower right. <br>Sequences of the sgRNAs and crRNAs used:tyr_Cas9_sgRNA6 GGUGCGCCGGAAACUACAUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUtyr_Cas9_sgRNA9 GGUGUUGGCGAACAUUGGCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUtyr_Cas9_sgRNA10 GGGUGUACGAUUUAUUCGUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUtyr_Cpf1_crRNA1 UAAUUUCUACUAAGUGUAGAUCAACCAAACCUGCCAGUGCGCCGtyr_Cpf1_crRNA2 UAAUUUCUACUAAGUGUAGAUUUCGUGUGGAUGCAUUAUUACGUtyr_Cpf1_crRNA3 UAAUUUCUACUAAGUGUAGAUCGCAUGAGUCGGCGGCGUUUCUG<br>This experiment reproduces Figure 1f/g of this preprint:https://www.biorxiv.org/content/early/2017/06/26/156125