# Enhancing homozygous genome editing through DNA recombination and biologic gates

> **NIH NIH R21** · ROCKEFELLER UNIVERSITY · 2022 · $211,875

## Abstract

Project Summary
With the discovery of the CRISPR/Cas system, human genome engineering has been revolutionized,
enabling a large number of genome editing operations. Our ability to control human genome editing
events therefore directly affects downstream biomedical research.
However, while the generation of biallelic knockouts has become a highly efficient process, our ability
to efficiently generate biallelic knock-ins is still severely limited. Key reasons for this are that in addition
to a Cas-mediated endonucleolytic cleavage event the following hurdles need to be overcome: First,
homologous recombination needs to occur on both alleles. Second with a single DNA repair template
it is difficult to reliably distinguish monoallelic from biallelic editing at a single cell level. Third, in cases
where two DNA repair templates are used, biallelically edited cells can be selected but the overall
efficiency is further reduced.
There is hence an urgent need for new technologies that enable efficient biallelic editing together with
powerful selection strategies. We hypothesize that this critical need can be addressed by synergistically
employing a Cas enzyme in conjunction with DNA recombination and BOOLEAN logic AND gates.
Based on these ideas we propose to develop a new genome editing platform, focusing on two specific
aims: (1) To facilitate biallelic genome editing with a single DNA repair template and DNA recombination
and (2) To develop a split reporter system that constitutes a BOOLEAN logic AND gate so that
biallelically edited clones can be selected with high efficiency.
These new approaches will significantly increase our ability to perform efficient biallelic human genome
editing and integrate biologic gates into this process, thus having widespread and immediate impact
across biomedical sciences.

## Key facts

- **NIH application ID:** 10490854
- **Project number:** 5R21GM143616-02
- **Recipient organization:** ROCKEFELLER UNIVERSITY
- **Principal Investigator:** Sebastian Klinge
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $211,875
- **Award type:** 5
- **Project period:** 2021-09-20 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10490854

## Citation

> US National Institutes of Health, RePORTER application 10490854, Enhancing homozygous genome editing through DNA recombination and biologic gates (5R21GM143616-02). Retrieved via AI Analytics 2026-06-14 from https://api.ai-analytics.org/grant/nih/10490854. Licensed CC0.

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