# Developing a CRISPR-free mammalian recombineering system

> **NIH NIH DP2** · SCRIPPS RESEARCH INSTITUTE, THE · 2024 · $1,638,033

## Abstract

Developing a CRISPR-free mammalian recombineering system
Project Summary / Abstract
The ability to manipulate large segments of DNA in mammalian cells is essential for research and therapeutics.
However, current programmable genome editing methods for creating kilobase-scale DNA manipulations
require cytotoxic double-strand breaks (DSBs) or cannot create all required manipulations1. To address this
major challenge, my laboratory at Scripps Research is developing a platform for mammalian recombineering
that enables programmable large DNA insertions, deletions, and substitutions without the introduction of DSBs
or DNA scars. Our preliminary data demonstrates that bacterial recombinases such as RecT from
Enterococcus faecium2 are sufficient to catalyze whole gene insertion in the human genome. In this proposal, I
describe the further characterization, optimization, and application of this system. First, we will fully
characterize the mechanism and capabilities of efmRecT to create different size insertions, deletions, and
substitutions across the genome using multiple methods of detection. Then we will engineer our
recombineering system using supplemental bacterial recombineering proteins, mammalian repair protein
inhibitors, and other manipulations informed by our mechanistic studies. We also aim to improve the efficiency
of efmRecT through directed evolution in bacteria and/or mammalian cells. Finally, we will demonstrate the
utility of our developed system by applying it to cell models of Gaucher Disease. The small size of RecT
enables facile biomolecule delivery as the RecT gene, homology donor, and regulatory sequences can be
encoded on a single recombinant AAV genome. As such, I am proposing an “all-in-one” AAV therapeutic
where a single recombinant AAV can correct any of the hundreds of mutations associated with Gaucher
Disease. If developed into a functional system for efficient genome editing, this method has the potential to
impact fields spanning research to the clinic, including genomic perturbations of complex cell lines and
organisms, methods for mammalian synthetic biology, and therapeutics.

## Key facts

- **NIH application ID:** 10910639
- **Project number:** 1DP2GM159183-01
- **Recipient organization:** SCRIPPS RESEARCH INSTITUTE, THE
- **Principal Investigator:** Shannon Marie Miller
- **Activity code:** DP2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $1,638,033
- **Award type:** 1
- **Project period:** 2024-09-01 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10910639

## Citation

> US National Institutes of Health, RePORTER application 10910639, Developing a CRISPR-free mammalian recombineering system (1DP2GM159183-01). Retrieved via AI Analytics 2026-08-01 from https://api.ai-analytics.org/grant/nih/10910639. Licensed CC0.

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