CRISPR functional genomics for target identification and therapeutics

CRISPR genome editing technologies have transformed both basic and biomedical research, enabling everything from therapeutic target identification and exploration of disease-relevant molecular pathways to gene therapy and the creation of engineered cellular therapeutics. To reach their full potential, however, critical improvements to CRISPR gene editing are needed, including the ability to be multiplexed, precise controllability, and broad applicability for functional genomics, particularly in mouse models of disease in vivo and disease-relevant human primary cells.

To bridge this gap, our laboratory is developing innovative technology platforms that facilitate genome editing and functional genetic screening both in vivo using mouse models and in primary cells. For instance, addressing the significant challenge of delivering CRISPR systems to primary cells simply, efficiently, and non-toxically, we have developed the engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system. This system enables rapid, robust, and gentle editing in primary cells from healthy donors and patients, achieving 85–100% editing efficiency after just a 30-minute incubation with the PAGE system. We have further expanded the PAGE system by integrating precision genome-editing tools. In addition, to meet the need for robust and inducible functional genetic screening in in vivo mouse models of cancer, we developed a series of chemically controlled, split-engineered base editors (seBEs) with optimized sgRNA design. This approach reveals loss-of-function and dominant negative mutations, including in complex in vivo screens that distinctively elicit non-cell autonomous cancer vulnerabilities. The seBE platform provides a powerful, inducible, and highly efficient method for systematically identifying key residues in functional genomics.