High-throughput creation and functional profiling of eukaryotic DNA sequence variant libraries using CRISPR/Cas9

ABSTRACT Construction of genetic variant libraries with phenotypic measurement is central to advancing today’s functional genomics, and remains a grand challenge. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions and insertions), along with methods for tracking their fitness en masse . We demonstrate the utility of our approach in performing focused analysis of hundreds of mutants of a single protein and in investigating the biological function of an entire family of poorly characterized genetic elements. Our platform allows fundamental biology questions to be investigated in a quick, easy and affordable manner.