Abstract
The CRISPR/Cas9 system has been adapted as an efficient genome editing tool in laboratory animals such as mice, rats, zebrafish and pigs. Here, we report that CRISPR/Cas9 mediated approach can efficiently induce monoallelic and biallelic gene knockout in goat primary fibroblasts. Four genes were disrupted simultaneously in goat fibroblasts by CRISPR/Cas9-mediated genome editing. The single-gene knockout fibroblasts were successfully used for somatic cell nuclear transfer (SCNT) and resulted in live-born goats harboring biallelic mutations. The CRISPR/Cas9 system represents a highly effective and facile platform for targeted editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications.
Highlights
Targeted genome editing technologies are crucial for basic biology research, development of animal models and improvement of animal traits for agriculture
Zinc finger nucleases [1,2], transcription activator-like effector nucleases [3,4] and homing meganucleases [5] have provided powerful tools to induce targeted mutations in the form of small insertions or deletions derived from DNA break repair of nonhomologous end joining (NHEJ) or homologous recombination
The plasmids targeting each gene were respectively transfected into goat fibroblasts, and their genome modification efficiency was determined at day 3 using restriction fragment length polymorphism (RFLP) assay
Summary
Targeted genome editing technologies are crucial for basic biology research, development of animal models and improvement of animal traits for agriculture. Zinc finger nucleases [1,2], transcription activator-like effector nucleases [3,4] and homing meganucleases [5] have provided powerful tools to induce targeted mutations in the form of small insertions or deletions derived from DNA break repair of nonhomologous end joining (NHEJ) or homologous recombination. These systems, require efficient design and time-consuming assembly of nuclease constructs for DNA targeting. These findings encouraged us to explore the possibility of establishing a Cas9/gRNA-based gene modification platform for large animals
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