We utilize metagenomic analysis of microbial communities across the tree of life to discover novel proteins for efficient genome editing. These systems are tested for function in vitro, heterologously in vivo, in situ microbial communities, and in eukaryotic cells.
The Doudna lab employs protein engineering and screening strategies to bestow CRISPR-Cas enzymes with improved and novel functionalities. In collaboration with Stanley Qi and the laboratories of Wendell Lim, Jonathan Weissman, and Adam Arkin, we pioneered the engineering of CRISPR-Cas proteins for new purposes by converting Cas9 into a tool for targeted control of gene expression, referred to as CRISPR interference (CRISPRi). In this platform, catalytically inactive Cas9 serves as an RNA-guided DNA binding protein to silence gene expression in a sequence-specific manner. In collaboration with the laboratory of David Savage, we have also developed a series of high-throughput mutant library construction, screening, and sequencing pipelines to probe CRISPR-Cas protein function and to isolate engineered variants with novel properties. In one example, we utilized domain insertion profiling with sequencing (DIP-seq) to identify an allosteric Cas9 switch, yielding an optimized Cas9 variant that is activated by the small molecule 4-hydroxytamoxifen. Recently, we used a similar large-scale protein engineering and screening platform to construct circularly permuted, viral protease-activated ProCas9s that orchestrate programmed cellular responses to pathogen-associated protease activity.
