# Programmable gene integration and cell engineering with CRISPR-directed integrases

> **NIH NIH R01** · BRIGHAM AND WOMEN'S HOSPITAL · 2024 · $664,371

## Abstract

Project Summary
Despite extraordinary advances in genome engineering, tools for precise and efficient gene correction across all
cell types and desired edits remain lacking. Current programmable DNA cleavage tools, such as CRISPR-Cas9,
rely on cellular DNA repair mechanisms, which are inefficient and do not function in post-mitotic cells. Thus,
genome editing still needs efficient, robust tools that can make a variety of specific DNA sequence alterations.
These tools could have broad applications across both basic biological discovery, allowing for new modalities of
screening, and therapeutics, including engineered cell therapies. The proposed work will address these needs
by combining computational discovery, biochemical characterization, and enzyme engineering to develop
integrase-based tools for programmable, multiplexed insertion of large genes in diverse cell types
independent of DNA repair. The discovery, characterization, and engineering of these new integrase proteins
will both build upon our deep history of CRISPR enzyme discovery, as well as draw from new, high-throughput
approaches to mine biological diversity. Complementary to the discovery of these new enzymes, we will combine
Cas9-based genome editing with integrase engineering to develop programmable, multiplexed genome
integration systems that do not depend on DNA repair mechanisms, allowing integration of large sequences in
any cell type. We will explore delivery mechanisms, including viruses, electroporation, and novel lipid
nanoparticle formulations to edit T cells and neurons. We will engineer aspects of the integrases, including
protein engineering and site mutagenesis, to boost activity of the system and screen many insertion sites to
develop design rules for the technology. Moreover, through studying orthogonal integrases sites we can develop
multiplexed versions of the insertion tool to edit up to three sites in a given cell with superior efficiency over other
tools. We will apply these multiplexed integrases to develop a new screening system, where tagging of multiple
genes can be used for determining protein interaction partners in high throughput. Our new integrase systems
will also be applied to the development of multiple-edited T-cells for improved immuno-oncology therapies. The
multiple technologies resulting from these discoveries and engineering efforts will overcome the limitations of
existing genome and epigenome engineering approaches and serve as a valuable resource for broader
biomedical research. Programmable gene integration with CRISPR-recruited integrases will allow for more
advanced genome engineering applications to be pursued in cells and in vivo, accelerating the pace of
biomedical research, enabling greater exploration of basic biological processes and disease mechanisms, and
promoting novel therapeutic developments.

## Key facts

- **NIH application ID:** 11097018
- **Project number:** 7R01EB031957-04
- **Recipient organization:** BRIGHAM AND WOMEN'S HOSPITAL
- **Principal Investigator:** Omar O Abudayyeh
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $664,371
- **Award type:** 7
- **Project period:** 2021-09-20 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11097018

## Citation

> US National Institutes of Health, RePORTER application 11097018, Programmable gene integration and cell engineering with CRISPR-directed integrases (7R01EB031957-04). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/11097018. Licensed CC0.

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