CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation

CRISPR-based genome editing tools have greatly facilitated the production of genetically engineered rep models. The CRISPR system is comprised of a single guide RNA complex to a Cas9 nuclease and can be programmed to target a sequence of interest within the genome to create a double stranded DNA break. Through the codelivery of a DNA repair template, this DNA break can be precisely restored along with the addition of any desired engineered DNA sequence through a process called Homology Directed Repair.

It is by this process that we are able to generate knock-in rat models with targeted DNA insertions or substitutions. Using this protocol, we present a modified approach using adeno-associated virus to deliver a DNA repair template to rat embryos, along with a subsequent delivery of CRISPR Cas9 complexes through two cell embryo electroporation. AAV serotypes 1 or 6 is packaged with an engineered DNA repair template designed to be integrated into the rat genome through the CRISPR-mediated Homology Directed Repair process.