Anti-CRISPR-mediated Acylation and Bioreversible Esterification for Precision Genome Editing

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PROJECT SUMMARY/ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems use RNA-guided proteins (e.g., Cas9, Cas12a) to cleave DNA. Cas9 and Cas12a share many similarities and have been repurposed for genome editing in human cells because of their programmability, simplicity, and efficiency, with applications in basic research and medicine. Cas12a is generally more efficient and specific than Cas9. However, since the concentration of Cas protein is much higher than that of the DNA target in genome editing, prolonged Cas12a activity also leads to off-target DNA cleavage, chromosomal translocations, and genotoxicity. Equipping Cas12a with an “on-off” switch can overcome these challenges by shortening Cas12a’s window of exposure and increasing its specificity—the on-target to off-target ratio of DNA cleavage. We developed a novel strategy that repurposes a CRISPR-Cas inhibitor protein (i.e., an anti-CRISPR) to selectively acylate or “cage” Cas12a with a small-molecule-removable group. We propose to deliver small molecules and anti- CRISPR proteins into human cells via fast, bioreversible, and in vivo-compatible means to activate and inactivate Cas12a, respectively, and increase its specificity. With the help of experts at the outstanding MIT and Broad Institute facilities, I will learn how to use liquid chromatography–tandem mass spectrometry (LC–MS/MS) to assess the selectivity of our novel Cas12a acylation strategy, and mammalian cell culture, high throughput confocal microscopy, next-generation sequencing, and computational analysis to assess anti-CRISPR delivery and Cas12a genome-editing specificity. Overall, this research will introduce the first approach for the site-selective and reversible acylation of Cas12a lysine residues and a new bioreversible esterification strategy for the traceless delivery of anti-CRISPR proteins that together will increase genome- editing specificity.