Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes

May 7, 2016The Journal of biological chemistry

Highly Efficient Mouse Genome Editing by Delivering CRISPR Proteins into Fertilized Eggs

AI simplified

CRISPR Gene Editing on OpenScience ↗PubMed ↗DOI ↗

Abstract

CRISPR-EZ achieves 100% efficiency in delivering genome-editing components into mouse zygotes.

Electroporation-based delivery of Cas9/sgRNA ribonucleoproteins improves embryo viability compared to traditional microinjection.
The method enables high-throughput genome editing in mouse embryos.
Generated editing schemes include indel mutations, point mutations, large deletions, and small insertions.
In targeting the tyrosinase gene, CRISPR-EZ resulted in 88% bi-allelic editing and 42% precise sequence modification.
CRISPR-EZ may serve as a simpler and more economical alternative to microinjection for in vivo genome editing.

AI simplified

The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals.

Full Text

Full text is available at the source.

View full text (XML) ↗View full text ↗

Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes

Abstract

Full Text

You found one interesting study. We’ll send the next 7.

what lands in your inbox each week:

Recent issues from the crispr gene editing brief