# Recombineering-based no-cleavage gene-editing toolkit for large-scale genome engineering and functional screening

> **NIH NIH R01** · STANFORD UNIVERSITY · 2024 · $417,908

## Abstract

Project Abstract
Recombineering-based no-cleavage gene-editing toolkit for large-scale genome engineering
and functional screening
Exemplified by the CRISPR-Cas9 system, gene-editing technology is a powerful collection of
tools for probing the hidden mechanisms of human diseases by understanding and controlling
the functions of human genome variants. Limitations of existing CRISPR tools stem from two
sources: 1) Cas9 cutting causes uncontrollable DNA damage at on/off-target sites, leading to
toxicity and stress response. Recent studies confirmed that cutting-induced damages lead to
significant gene expression changes and enrichment of p53-mutant cells, thus confounding
some CRISPR screens; 2) CRISPR enzymes do not repair the target DNA, thus relying on
endogenous DNA repair to complete editing. This results in low efficiency and high variability for
Cas9-mediated homology-directed repair (HDR) across cell types and models. To overcome
these limitations, we have identified a recombineering-based gene-editing tool, termed RecE/T-
induced Editing via Designer-Cas9-Initiated Targeting (REDIT). REDIT uses deactivated Cas9
(dCas9) and generates minimal DNA break and near-zero toxicity. REDIT uses phage
recombineering proteins RecE/T for gene-editing, bypassing the dependence on endogenous
repair mechanisms. Our proof-of-concept demonstration showed that REDIT achieved efficient
kb-scale editing without DNA cutting. We will focus on technology development and validation
with well-characterized models using gold-standard assays. The proposed RecE/T-like
recombineering proteins present new opportunities as they promote strand invasion/exchange
without cleavage when genome sites become transiently accessible via dCas9 DNA-unwinding.
Our goal is to develop a safe, scalable toolkit with up to 80% HDR efficiency for kilobase gene-
editing and pooled knock-in screening.

## Key facts

- **NIH application ID:** 10833581
- **Project number:** 5R01GM141627-04
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Le Cong
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $417,908
- **Award type:** 5
- **Project period:** 2021-08-10 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10833581

## Citation

> US National Institutes of Health, RePORTER application 10833581, Recombineering-based no-cleavage gene-editing toolkit for large-scale genome engineering and functional screening (5R01GM141627-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10833581. Licensed CC0.

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