Unraveling Molecular Mechanisms for Enhanced Precision Genome Editing

Nearly half of human genetic diseases are caused by point mutations. As such, there is an urgent need for advanced precision genome editing tools capable of efficiently and accurately correcting these mutations. Current CRISPR-Cas-based technologies, including DNA base editors (ABEs) and prime editors (PEs), are designed to make targeted single nucleotide changes without introducing double- stranded breaks, relying instead on the homology-directed repair pathway.

However, despite their potential, these tools still face limitations in cellular applications, such as off-target DNA and RNA editing, low editing efficiency, and unintended bystander editing of nucleotides adjacent to the target site. These issues can result in undesirable genomic changes that compromise editing precision.

At CTVR, we are working to overcome these challenges by gaining a deeper understanding of the molecular mechanisms underlying precision genome editing tools. Our research employs cutting-edge techniques such as cryo-electron microscopy (cryoEM), molecular dynamics (MD) simulations, and comprehensive biophysical and biochemical characterization to refine these technologies. In our recent work, we uncovered important insights into the mechanism of ABE8e, a widely used DNA base editor. We demonstrated that ABE8e forms dimers through the dimerization of its deaminase domains (TadA), and this dimerization is essential for efficient DNA base editing. We found that while one TadA subunit catalyzes the deamination reaction, the second subunit—acting as the "docking" subunit—plays a crucial role in building the DNA binding pocket and interacts specifically with the RuvC domain of Cas9.

These findings are critical for improving the precision and efficiency of ABEs. We are exploring strategies to engineer the TadA-Cas9 interaction interface, ensuring better control over the movement of deaminase and Cas9, thereby minimizing bystander editing. This research paves the way for more accurate and efficient genome editing technologies, particularly when pairing smaller Cas effectors with these precision editing tools.