# Leveraging Programmable Integrases for Human Genome Engineering

> **NIH NIH DP2** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2020 · $2,430,000

## Abstract

PROJECT SUMMARY
 LEVERAGING PROGRAMMABLE INTEGRASES FOR HUMAN GENOME ENGINEERING
The genetic engineering toolbox comprises a diverse array of molecular machineries for genome manipulation,
and DNA insertion methods have arguably had the largest impact on biomedical research. Gene knock-ins are
used in the clinic to treat genetic diseases and cancer, in industry to manufacture biologics, in agriculture to
improve crops, and in research to generate models of human disease, among many other uses. These
applications generally depend on either random integration mediated by viruses and transposases, or site-
specific integration mediated by homologous recombination and gene editing. The former category exhibits high
efficiency but little specificity, whereas the latter category is inherently precise but reliant on cellular factors and
thus ineffective. Only recently has a new molecular functionality been discovered that is both fully autonomous
and also highly accurate: programmable integrases directed by CRISPR RNAs.
 CRISPR systems have revolutionized biology over the past decade because of how easily one can program
CRISPR-associated nucleases with guide RNAs to introduce DNA double-strand breaks, the precursor to DNA
repair. Whereas the inability to easily redesign engineered nucleases previously stalled gene-editing technology,
the discovery of RNA-guided DNA targeting eliminated this critical bottleneck. A similar bottleneck for engineered
integrases is now ready for elimination.
 My central vision is to develop programmable, RNA-guided integrases as a powerful new platform
technology for human genome engineering. Building on our recent work that deciphered sequence determinants
of this technology in bacteria, as well as parallel studies that expanded the CRISPR–Cas subtypes that function
robustly in mammalian cells, we will embark upon a systematic effort to build the capabilities for employing these
multi-subunit integrases in eukaryotic cells. We will then develop the first tools for performing simultaneous,
multiplexed DNA insertion events across thousands of distinct genomic target sites using guide RNA libraries.
This approach will enable us to probe fundamental questions regarding the role of noncoding elements such as
enhancers and insulators in regulating gene expression. Furthermore, we will harness orthogonal integrases to
execute highly programmed translocation events and study the role of complex genome rearrangements in
disease and cancer. Our studies will contribute powerful new tools to the genetic engineering toolbox and open
the door to genomic manipulations that are inaccessible with any other experimental approach.

## Key facts

- **NIH application ID:** 10002492
- **Project number:** 1DP2HG011650-01
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** Samuel Henry Sternberg
- **Activity code:** DP2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $2,430,000
- **Award type:** 1
- **Project period:** 2020-09-09 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10002492

## Citation

> US National Institutes of Health, RePORTER application 10002492, Leveraging Programmable Integrases for Human Genome Engineering (1DP2HG011650-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10002492. Licensed CC0.

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