# Engineering Efficient and Controllable Base Editors

> **NIH NIH R01** · UNIVERSITY OF PENNSYLVANIA · 2024 · $437,938

## Abstract

This proposal aims to manipulate DNA deaminase enzymes to generate hyperactive and controllable base
editors that can be targeted for precise gene editing. Base editing of the immunoglobulin locus by AID, the
ancestral member of the AID/APOBEC family of cytosine deaminase enzymes, normally initiates maturation of
antibody responses in B-cells, while APOBECs provide protection against retroviruses. Out of their physiological
context, when DNA deaminases are directed by catalytically-impaired CRIPSR/Cas proteins, their base editing
activity can be used to introduce targeted mutations at a desired genomic locus. While this system offers a
potentially powerful means to edit the genome for biological or therapeutic purposes, base editors have two
barriers that limit their broader application in basic and translational research. First, DNA deaminases have
naturally evolved to be constrained enzymes with low overall catalytic activity, as hyperactivation is associated
with increased oncogenic mutations. Second, when dysregulated, AID/APOBECs are known to act outside of
their targets, promoting cancer mutagenesis, chromosomal translocations, and resistance to chemotherapy.
Given that natural regulatory constraints on DNA deaminases are lost in base editor complexes, these constructs
pose similar risks to the genome. In this proposal, we harness our extensive knowledge of the mechanism,
structure and function of deaminase enzymes in order to overcome these challenges. For one, hyperactive
deaminases have been generated to overcome the naturally attenuated activity, and we will exploit these variants
to evaluate the hypothesis that increasing the deamination rate can improve the efficiency of the base editing
reaction, while simultaneously improving precision. Second, we have devised split deaminases that can only be
reconstituted at the targeted locus under the control of a small molecule. This strategy newly offers
spatiotemporal control, a critical requirement that will facilitate the use of base editors in the lab and is essential
to therapeutic applications in patients. Given the wide range of potential uses for base editors, we will
demonstrate the importance of efficiency and control broadly across diverse genomic sites, and then specifically
by generating enhanced chimeric antigen receptor expressing T (CAR-T) cells as a model system. The tools
developed here will globally advance deaminases as base editors and will readily translate to other innovations
in CRISPR/Cas proteins, and to genome engineering more generally.

## Key facts

- **NIH application ID:** 10830957
- **Project number:** 5R01GM138908-04
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Rahul Manu Kohli
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $437,938
- **Award type:** 5
- **Project period:** 2021-05-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10830957

## Citation

> US National Institutes of Health, RePORTER application 10830957, Engineering Efficient and Controllable Base Editors (5R01GM138908-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10830957. Licensed CC0.

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