Zebrafish CRISPR-Cas9 Knockout Generation

Gene Knockouts were generated as described previously [72 (link)]. gRNA target sites were identified using the Zhang lab gRNA design tool (gRNA sequencing and target sites listed in S3 Fig). The CRISPR gRNA sequences were cloned into pDR274 (Addgene NO. 42250). The Cas9 mRNA was transcribed from pT3TS-nCas9n (Addgene No. 46757) [73 (link)]. After cloning specific target plasmids/guides into pCS2 variant vector, mRNA was generated by in vitro transcription of NotI-HF linearized DNA using the Invitrogen mMESSAGE mMACHINE SP6 Transcription Kit (Fisher Scientific AM1340) and purified with the MEGAclear Transcription Clean Up Kit (Fisher Scientific AM1908). 1-2nl of sgRNA/Cas9 mRNA was microinjected into the yolk of one-cell-stage wild-type zebrafish embryos. For indel efficiency evaluation, genomic DNA was extracted from ~24 3dpf injected embryos and evaluated with HRM (see below). The remaining embryos (F0s) from the same clutches were raised. Out-of-frame indels identified in F1 progeny with Sanger sequencing were maintained and propagated. To “clean up” genetic background all lines were bred at least 2 generations to the wild-type strain AB.