# Optimization of donor structure enhances the generation of cloned goats with high expression of human butyrylcholinesterase by CRISPR/Cas9

## TL;DR

Scientists improved CRISPR editing in goats to produce high levels of a human enzyme that can protect against nerve agents.

## Contribution

A novel donor structure optimization strategy significantly enhances HDR efficiency in large animal genome editing.

## Key findings

- Reverse-oriented donor structures significantly improved HDR efficiency compared to forward designs.
- Cloned goats expressed high levels of human butyrylcholinesterase in their skin.
- Edited cells showed increased resistance to organophosphorus pesticides.

## Abstract

Human butyrylcholinesterase (hBChE) is a promising bioscavenger against organophosphorus (OP) nerve agents and pesticides. However, low homology-directed repair (HDR) efficiency in CRISPR/Cas9-mediated genome editing limits precise transgene integration in large animals.

To improve HDR, we optimized donor structure for targeted integration of hBChE into the goat FGF5 locus using CRISPR/Cas9. Correctly edited goat fibroblast clones were identified by PCR and sequencing. A homozygous clone with reverse-oriented integration was used as a donor for somatic cell nuclear transfer (SCNT), followed by embryo transfer. Offspring were analyzed for genomic integration and transgene expression.

Reverse-oriented donors significantly enhanced HDR efficiency compared to forward designs, with validation at the pig RAG1 locus. Edited cells stably expressed recombinant hBChE (rhBChE) and showed increased resistance to OP pesticides. SCNT produced a cloned goat expressing high rhBChE levels in the skin.

Optimizing donor structure improves precise genome editing efficiency and enables robust generation of transgenic goats. This strategy advances CRISPR/Cas9-based bioreactor development for scalable production of therapeutic proteins.