Scalable characterization of enzymes to inform engineering of gene editing technologies

Abstract

The discovery of RNA-guided CRISPR-Cas nucleases has made targeted genome editing accessible for diverse research and clinical applications. Simple reprogramming of the guide RNA (gRNA) directs Cas proteins to a new DNA target sequence. The Cas system is now the backbone of next-generation editors which catalytically inactivate the Cas nuclease domains but use the search function of the protein to localize other DNA modifying enzymes to specified target sequences. For example, base editors (BEs) utilize a wide range of deaminase enzymes to install single nucleotide edits. DNA writing technologies, such prime editors (PEs) and click editors (CEs), fuse polymerases and nucleic acid templates to Cas to write in specified edits. As the gene editing field rapidly expands, the question changes from what technology should we build towards what technology should we apply? To better inform BE design, we build new methods for rapid and scalable characterization of BE kinetics, specificity, and sequence preference to make informed decisions on BE engineering and applications. Additionally, we explore molecular techniques to observe unintentional editing by a nascent gene editing approach called click editing. Together, this work advances the systematic evaluation of editors and contributes practical frameworks for identifying unintended editing behavior of emerging editors, bridging the gap between rapid technological development and the evaluation necessary for safe and effective application of gene editing tools.