Figure 2. Strategy to generate NRG Fah knockout mice.

(a) Schematic illustration of IVF and pronuclear microinjection. Female NRG mice are superovulated with PMSG and hCG followed by oocyte collection. Sperm is collected from male NRG mice. The oocytes and sperm are incubated to generate fertilized eggs and embryos, which are then microinjected with gRNA, Cas9 mRNA and ssDNA in the pronucleus. The injected embryos are then transferred into pseudopregnant surrogate mothers. Mouse pups are genotyped. (b) Mechanism of gRNA, Cas9 mRNA, and ssDNA mediated Fah gene knockout. Cas9 mRNA is translated into Cas9 protein after microinjection. The Cas9/gRNA complex binds the genomic DNA and generates DSBs. The ssDNA contains homologous sequence spanning the double strand break sites with ~50 bp on each side, two stop codons and a BamHI site. The ssDNA can be used as a template for homologous recombination to introduce the stop codons and BamHI site. The mouse is drawn by the authors (F.L. and L.S.), using Adobe Photoshop and Adobe Illustrator.