Abstract 1013 Investigating the CRISPR-Cas12a mechanism to design hyper-efficient genome editors

Base editors (BEs) are fusion proteins that harness the power of RNA-programmed CRISPR-Cas proteins and DNA deaminases, allowing precise editing of the genome. Unlike the more widely used Cas9 proteins that induce single-locus edits, proteins from the Cas12a family can target and transcriptionally activate up to 15 sites using a single multiplexed RNA transcript. Therefore, Cas12a BEs can edit multiple loci in a programmable manner using the same protein, greatly expanding the potential of polygenic genetic screens to reveal novel genetic pathways. Additionally, multiplex editing can be harnessed to treat more than 50% of human genetic diseases caused by missense mutations. However, the capacity of Cas12a BEs editing has yet to be fully realized. Cas12a BE efficiencies (average 35%) remain low when compared to Cas9 BEs (typically >90%). Thus, higher-efficiency editors must be developed to capitalize on the unique multiplexing potential of Cas12a BEs. Here, we aim to identify the rate-limiting step of Cas12a BEs and improve their genomic editing efficiencies. Since little is known about the mechanism by which Cas12a BEs catalyze edits, we propose a three-step model to develop a clear framework for investigating BE kinetics. The first step is DNA binding, in which the Cas component of the BE scans dsDNA to bind a specific sequence known as the protospacer adjacent motif (PAM) and the complementary DNA sequence to the user-defined CRISPR RNA (crRNA). We investigate Cas-DNA binding by engineering stronger DNA binding Cas variants. In the second step, the tethered deaminase catalyzes C>T or A>T conversions on the non-crRNA bound DNA strand. We examine deaminase activity by assessing the editing efficiency of Cas12a proteins fused to a series of hyperactive deaminases. In the third step, the BE detaches, the DNA bubble reseals, and replication to lock in the lesion competes with endogenous DNA repair mechanisms. Unlike Cas9 counterparts, Cas12a BEs do not nick DNA to direct DNA repair to the unedited strand. We study the relationship between DNA repair and editing by testing Cas12a BEs in cell lines with knocked-out DNA repair enzymes that could revert edits. By perturbing each step and manipulating the fundamental mechanisms of BEs, we aim to design the next generation of precise and hyper-efficient Cas12 biotechnological tools. NIGMS-R01-GM138908 to Dr. Rahul Kohli Vagelos MLS Fund Frances Velay Fellowship by Panaphil and Uphill foundations