Abstract One of the best methods for identifying novel genes and mutations affecting embryonic development is forward genetic screening. However, these screens require a large number of animals to be screened to identify a single novel mutant due to the inefficiency of chemical mutagenesis. Our current R15 finalized a method for enzymatic generation of CRISPR libraries and used the method to generate sgRNAs targeting all of, and only, the genes expressed in the developing zebrafish heart. The sgRNA templates were then divided into eight groups of ten, transcribed, and injected into single-cell zebrafish embryos. Amazingly, all eight pools showed clear morphological heart phenotypes. Thus, we are applying for this renewal grant to continue work on the screen. We have identified a gene from the first round of the screen, sytl5. We will describe this gene’s role in heart development, identify other mutated genes in our screen, and characterize their phenotypes in detail. We also propose to optimize screen parameters to maximize our efficiency and to apply our CRISPR library to a tissue-specific gene editing screen. In addition to the novel mutants identified in the screen, this method will be able to be adapted to any tissue in any species, reducing the resources needed to conduct forward genetic screens and increase the speed of gene discovery in a number of species and developmental processes.