PROJECT SUMMARY Forward genetic screening is a remarkably powerful research technique, but has been widely abandoned. Instead, modern genetics research relies heavily on reverse genetics approaches, revolutionized and made more accessible via technological advances such as CRISPR/Cas9. As the vast majority of mutations are recessive, CRISPR/Cas9 has enabled researchers to rapidly produce mutant phenotypes in reverse genetic studies by simultaneously disrupting both alleles of a known gene of interest. By contrast, the random mutagenesis paradigms used for forward genetic screening have remained relatively unchanged for the past 40 years, and still rely on generations of inbreeding to homozygose mutant alleles in animal models. No mutagenesis technique has ever been developed that is both random and biallelic. Here we propose to leverage modern genome-editing approaches to efficiently introduce mutations that are both random and biallelic, revolutionizing the field of forward genetics by supporting a new generation of unbiased interrogations of gene function across entire genomes. Our proposed paradigm achieves random biallelic mutagenesis using a two-step process. First, a Prime Editor enzyme (PE) stochastically reprograms the DNA-binding domain of a single-chain TALEN (scTALEN). Prime editing occurs cell-independently; thus a culture with millions of cells would generate millions of unique DNA- binding domains, each recruiting the scTALEN to a distinct genomic target site in each individual cell. Second, the scTALEN induces biallelic mutations at its newly programmed target site. The end result is a large collection of cells or embryos that each harbor unique biallelic mutations which can then be screened for phenotypes of interest. Most importantly, this process can be deployed in virtually any model organism from bacteria to mammals. Using the larval zebrafish system, we will illustrate how this approach shortens the mutagenesis timeline for forward genetic screening from approximately one year down to just a few weeks. We will develop our proposed paradigm, called TERM (TALEN Editing for Random Mutagenesis) by: 1) evaluating biallelic mutagenesis efficiency and toxicity of different scTALEN variants; 2) optimizing the prime-editing reaction to achieve efficient randomization of scTALEN targets; and 3) using TERM to perform forward genetic screens in vitro and in vivo. By breaking down the longstanding technical barriers to saturation mutagenesis screening, TERM will open doors to one of the most powerful techniques in genetic research. Specifically, our approach will help to characterize the ~80% of vertebrate genes that remain understudied, counteract longstanding bias towards studying coding regions, facilitate drug target identification and highlight new therapeutic targets for disease treatments, and expand genome-wide genetic analysis to models beyond cell-culture systems.