Engineering Controllable And Efficient Base Editors By Targeted Manipulation Of Dna Deaminases
Date of Award
Doctor of Philosophy (PhD)
Biochemistry & Molecular Biophysics
Rahul M. Kohli
Base editors (BEs) combine DNA deaminase mutator activity with CRISPR-Cas localization to create targeted point mutations in genomic DNA. Current approaches with BEs have enabled single-base alterations for several applications, including modeling and correction of disease alleles, crop engineering, and gene diversification. However, two major challenges limit their applicability: (1) moderate editing efficiency and (2) off-target mutagenesis of DNA and RNA. Here, we leverage our mechanistic knowledge into DNA deaminases to separately address both challenges. For one, nature has evolved DNA deaminases with suboptimal activity to achieve their role in immunity and minimize genomic instability. By deriving and characterizing hyperactive deaminases, I revealed intrinsic deaminase activity as a rate-limiting step in the base-editing reaction. Interestingly, hyperactive deaminases also had a broadened activity window, revealing a tradeoff between efficiency and precision. By harnessing their broad activity and skewing repair, we developed novel diversifying BEs that generate simultaneous C>T and G>A mutations efficiently over an expanded editing window of more than 65 bp. Second, DNA deaminases are highly regulated to achieve purposeful mutagenesis in physiological settings. Inspired by nature, I aimed to build regulatory control into the activity of DNA deaminases by splitting the enzyme into two inactive fragments, whose reapproximation reconstitute activity. This finding allowed me to develop small-molecule-inducible split-engineered base editors, which show decreased off-target editing when compared to intact BEs and newly enable temporal control over precise genome editing. Third, understanding the mechanistic basis for the preferential targeting of deaminases for DNA over RNA could provide means for engineering variants with decreased reactivity towards RNA. Thus, we developed biochemical assays to characterize the activity of AID/APOBEC enzymes on both substrates. Focusing on APOBEC3A, we establish the target base as a major determinant of selectivity and demonstrate that although overall deamination is greatly reduced in RNA, there is a strong selectivity for idealized substrates. Altogether, my results offer mechanistic insights into the incorporation of DNA deaminases in BEs, facilitating the development of enhanced base editing tools for diverse applications.
Berrios Adorno, Kiara Nicole, "Engineering Controllable And Efficient Base Editors By Targeted Manipulation Of Dna Deaminases" (2022). Publicly Accessible Penn Dissertations. 5522.