# Generating golden Syrian hamsters with conditional alleles via zygote microinjection of CRISPR/Cas9

## TL;DR

Researchers created the first conditional knockout model in golden Syrian hamsters using CRISPR/Cas9, enabling future studies on gene function and disease.

## Contribution

First successful conditional knockout model in golden Syrian hamsters using CRISPR/Cas9 and loxP-flanked gene editing.

## Key findings

- Conditional alleles of the ApoF gene were generated with up to 27% efficiency in hamster embryos.
- Floxed alleles were transmitted through the germline and validated for Cre-mediated recombination in the heart and brain.
- This model provides a new genetic tool for studying gene function and disease in hamsters.

## Abstract

The golden Syrian hamster is a valuable animal model for studying carcinogenesis, metabolic disorders, cardiovascular diseases, and viral infections due to its biological and pathological similarities to humans. However, the development of genetically engineered hamsters has lagged behind that of mice and rats, largely because of an embryonic development block at the two‐cell stage in vitro. Although CRISPR/Cas9‐mediated gene knockout has been achieved in hamsters, precise DNA fragment insertion or conditional knockout (cKO) models have not previously been reported, likely due to technical limitations in embryo manipulation and insufficient efficiency of homology‐directed repair (HDR).

In this study, we generated conditional alleles of the ApoF gene in golden Syrian hamsters. A two‐cut strategy was applied using Cas9 protein, two sgRNAs, and a single donor plasmid containing exon 2 flanked by loxP sites and two ~0.8 kb homology arms. A mixture of Cas9 protein, sgRNAs, and the donor plasmid was microinjected into the pronuclei of one‐cell stage hamster embryos.

The efficiency of CRISPR/Cas9‐mediated loxP knock‐in reached up to 27%, and the genetically modified floxed alleles were successfully transmitted through the germline. The functionality of the inserted loxP sites was validated by in vivo Cre‐mediated recombination following local administration of AAV vectors, including AAV‐cTnT‐Cre in the heart and AAV‐CMV‐Cre in the brain.

To our knowledge, this work represents the first successful establishment of a conditional knockout model in the golden Syrian hamster, providing a valuable tool for mechanistic studies of gene function and disease modeling.

We established the first conditional knockout (cKO) model in the golden Syrian hamster by CRISPR/Cas9‐mediated genome editing. Cas9 protein, two sgRNAs, and a donor plasmid carrying loxP‐flanked exon 2 of the ApoF gene were microinjected into one‐cell embryos. The floxed allele was efficiently generated (up to 27%) and transmitted through the germline. Functionality of the loxP sites was confirmed by Cre recombination in vivo after AAV‐Cre delivery to the heart and brain. This work provides a valuable genetic tool for mechanistic studies and disease modeling in hamsters.