# ADVANCING GENE-EDITING NUCLEASES FOR DIVERSE ZEBRAFISH APPLICATIONS

> **NIH NIH R01** · UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH · 2024 · $461,081

## Abstract

Summary
 CRISPR-Cas9 and related technologies have dramatically transformed our ability to manipulate the
genomes of countless organisms, generate disease models, rapidly discover the functions of genes, and
create new treatments for genetic disorders. Nevertheless, the full potential of the field has yet to be realized.
Over the past fifteen years, the Peterson and Yeh labs have collaborated in the development of genome
editing tools and have contributed to several key advances, including the first use of CRISPR-Cas9 to modify
the genome of any animal, the first use of prime editing in zebrafish, and the first technology for high-
throughput CRISPR screening in a vertebrate. During the current funding period, we have successfully
completed all three of our aims and produced new technologies, TICIT and MIC-Drop, that raise exciting new
opportunities for further development and application. In this competitive renewal application, we propose to
expand on TICIT and MIC-Drop, developing several useful new gene-editing approaches.
 Aim 1 builds upon Targeted Integration by CRISPR-Cas9 and Integrase Technologies (TICIT), which
utilizes the site-specific DNA recombinase – phiC31 integrase – to insert DNA into genomic target sites that
have been pre-specified by CRISPR-Cas9 modification. Unlike traditional transgenic methods which lead to
random genome insertion, TICIT enables precise integration of plasmids into the genome at prespecified loci,
avoiding inadvertent gene disruption and positional effects. Building upon TICIT, we propose now to expand
the utility of integrases for site-specific genome editing. In the renewal application, we propose new
technologies to make TICIT more efficient and permit combinatorial uses of multiple integrases in various safe-
harbor genomic loci. Additionally, we will use the optimized TICIT platform to develop high-throughput
screening systems for isolating synthetic promoters with enhanced cell-state or cell-type specificity.
 Aim 2 builds upon Multiplexed, Intermixed CRISPR Droplets (MIC-Drop), which combines multiplexed
CRISPR sgRNAs, Cas9 protein, and DNA barcodes into nanoliter-volume microfluidic droplets that can then be
injected from a single needle into thousands of zebrafish. MIC-Drop enables rapid and efficient disruption of
hundreds or thousands of genes, and after identification of phenotypes of interest, the causative gene can be
quickly identified by barcode recovery. Building upon MIC-Drop, we propose now to expand the utility of
MIC-Drop for molecular phenotyping. Previously, we have used visual inspection to identify zebrafish
mutants with interesting phenotypes, followed by PCR-based recovery of the DNA barcodes. We now plan to
combine MIC-Drop technology with powerful molecular phenotyping tools, such as single-cell RNA sequencing
(scRNAseq) and metabolomics. In this aim, we will develop new methods, barcodes, and workflows that will
enable efficient gene disruption and molecular phenotyping at un...

## Key facts

- **NIH application ID:** 10912042
- **Project number:** 5R01GM134069-06
- **Recipient organization:** UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH
- **Principal Investigator:** RANDALL T PETERSON
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $461,081
- **Award type:** 5
- **Project period:** 2019-09-20 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10912042

## Citation

> US National Institutes of Health, RePORTER application 10912042, ADVANCING GENE-EDITING NUCLEASES FOR DIVERSE ZEBRAFISH APPLICATIONS (5R01GM134069-06). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10912042. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*