# Development of a novel genome targeting technology using rational design and continuous evolution.

> **NIH NIH R21** · UNIVERSITY OF HAWAII AT MANOA · 2020 · $212,250

## Abstract

PROJECT SUMMARY
Despite great advances in genome editing technology, few clinical gene delivery therapeutics currently exist.
Concerns over unwanted genome alterations that can be caused by current technologies have tempered
excitement and delayed translational progress. New tools are needed to allow researchers and clinicians to
direct DNA to a single sequence without concern for off-target disruption. Safer technologies could broaden
genomic therapy beyond terminal disorders and warrant the treatment of increasing numbers of candidate
diseases. Unlike current gene targeting approaches that act passively, recombinase proteins are capable of
actively inserting DNA into the genome. Several recombinases are highly efficient at genomic integration,
although no recombinase is single-site specific. In this proposal, the exquisite site specificity of the CRISPR
Cas9 system will be leveraged with the robust efficiency of actively integrating recombinases. Directed
evolution is capable of imparting entirely new activities on proteins. New evolution technologies are orders of
magnitude faster at improving proteins and could be used simultaneously to increase both DNA binding
specificity and DNA integration efficiency. There exists a need for safer technologies capable of efficiently
delivering therapeutic DNA to desired sequences in the genome. The long-term goal is to generate a targeting
technology capable of efficiently inserting DNA at a single location in the genome without unwanted off-target
alterations. The overall objective of this application is to demonstrate the proof-of-concept that rational design
and directed evolution are capable of improving this specificity. The central hypothesis is that modifications to a
recombinase will improve the targeting specificity to sequences in the human genome. This hypothesis has
been formulated based on literature reports and preliminary experiments from the applicant's laboratories
demonstrating that fusion proteins consisting of custom DNA-binding proteins fused to the piggyBac
transposase are capable of targeting desired locations in the genome. A key innovation in this proposal is the
use of a rapid directed evolution technique called phage-assisted continuous evolution (PACE) that is capable
of performing dozens of cycles of evolution in a single day. The central hypothesis will be tested by pursuing
two specific aims: 1) Improve genome targeting specificity of recombinases using rational design; and 2)
Improve integration efficiency and specificity of recombinases using directed evolution. This proposal is
significant because it develops a new tool capable of safely and efficiently directing therapeutic genes to
desired locations in the genome. This technology applies to both preclinical research using gene targeting as
well as potential treatments of any disease requiring gene replacement. The proposed innovative
modifications, including a novel directed evolution approach, have not been previously ...

## Key facts

- **NIH application ID:** 9936398
- **Project number:** 5R21GM132779-02
- **Recipient organization:** UNIVERSITY OF HAWAII AT MANOA
- **Principal Investigator:** Jesse Bruce Owens
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $212,250
- **Award type:** 5
- **Project period:** 2019-06-01 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9936398

## Citation

> US National Institutes of Health, RePORTER application 9936398, Development of a novel genome targeting technology using rational design and continuous evolution. (5R21GM132779-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9936398. Licensed CC0.

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