Zebrafish Cre/loxregulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation

The ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/loxsystem has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/loxregulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron inhdac1, rbbp4, andrb1. Active, gene off orientationhdac1-UFlip-Offandrb1-UFlip-Offintegration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientationrbbp4-UFlip-Onandrb1-UFlip-Onintegration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs ofloxPandlox2272sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstraterbbp4-UFlip-Onandrb1-UFlip-Ongene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP inrbbp4-UFlip-Off, or 2A-KalTA4 inrb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/loxresponsive conditional gene alleles to advance zebrafish Cre recombinase genetics.