# Optimization of homology-directed repair in zebrafish

> **NIH NIH R21** · UNIV OF MASSACHUSETTS MED SCH WORCESTER · 2021 · $251,250

## Abstract

PROJECT SUMMARY
Genome editing with site-specific nucleases has facilitated application of reverse genetic approaches
to the zebrafish model. The recent advent of highly specific and active RNA-guided nucleases have
reduced cost and technical hurdles for genome editing, allowing reverse genetics to be applied in
nearly all zebrafish laboratories. In the majority of published applications, these tools have been used
to introduce targeted insertions or deletions (indels) by virtue of DNA repair of double strand breaks
via non-homologous end-joining (NHEJ) following cleavage. A more limited number of studies have
utilized homology-directed repair (HDR) with a DNA donor together with a sequence-specific
nuclease to introduce sequences of interest into the zebrafish genome. While researchers have had
success in this regard, rates of HDR-mediated repair events are consistently much lower than indel
frequencies from NHEJ. Furthermore, many sites remain recalcitrant to knock-in at acceptable rates.
This issue raises a significant barrier to the efficient production of desired knock-in alleles in the
zebrafish and the widespread use of this technique in the field. Therefore, additional studies are
needed to improve HDR for precise genome editing in zebrafish. In the proposed studies, we will
optimize conditions for targeted knock-in to allow efficient precise genome editing in the zebrafish. In
Aim 1, we will expand our application of novel parameters, identified through preliminary studies
showing they that drastically improve knock-in rates, to a set of 10 loci to confirm that they are
generally applicable. In all cases, we will apply definitive and accurate deep sequencing-based
analysis of somatic editing rates, along with assessment of germline transmission to identify optimal
parameters that are generalizable across all targets. In Aim 2, we will use optimized knock-in
parameters to generate a series of zebrafish bearing "Biotagged" endogenous loci to enable cell type-
specific enhancer and transcriptome analysis. These lines, together with the optimized parameters for
precise genome editing, will serve as valuable resources for the zebrafish community.

## Key facts

- **NIH application ID:** 10213866
- **Project number:** 5R21OD030004-02
- **Recipient organization:** UNIV OF MASSACHUSETTS MED SCH WORCESTER
- **Principal Investigator:** NATHAN D LAWSON
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $251,250
- **Award type:** 5
- **Project period:** 2020-07-15 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10213866

## Citation

> US National Institutes of Health, RePORTER application 10213866, Optimization of homology-directed repair in zebrafish (5R21OD030004-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10213866. Licensed CC0.

---

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