CRISPR sites, donors, primers, and Ct values.

<div> Precise regulation of gene expression is essential to understand a wide range of biological processes. Control over gene expression can be achieved using site-directed recombinases and endonucleases whose efficiency is variable and dependent on the genomic context. Here, we develop a self-cleaving ribozyme-based tool to control mRNA levels of endogenous targets in zebrafish. Using an <i>in vivo</i> reporter strategy, we first show that inserting the <i>T3H48</i> self-cleaving ribozyme in an intron enables rapid pre-mRNA cleavage, with up to 20-fold reduction in expression, and that this ribozyme displays superior activity compared with other ribozymes. We then inserted the <i>T3H48</i> ribozyme in the second intron of the <i>albino</i> gene using a CRISPR/Cas9 strategy and observed a pigmentation phenotype similar to that in the mutant. Using a base-editing strategy to inactivate the ribozyme, we also show that this phenotype is reversible, illustrating the specificity of the approach. In addition, we generated a Flippase- and Cre-activatable version of the <i>T3H48</i> ribozyme, called RiboFlip, to control the mRNA levels of the <i>albino</i> gene. RiboFlip activation induced mRNA knockdown and also recapitulated the <i>albino</i> mutant phenotype. Furthermore, we show that a Cre- and Dre-controllable Gal4/UAS reporter in the RiboFlip cassette can label knocked-down cells independently of the expression of the target gene. Altogether, we introduce the RiboFlip cassette as a flexible tool to control endogenous gene expression in a vertebrate model and as an alternative to existing conditional knockdown strategies. </div>