A Surgical Method for Oocyte Injection and CRISPR-Cas9 Mutagenesis in Anolis Lizards

Squamates, the taxon that comprises lizards and snakes, are a diverse assemblage of reptiles represented by more than 11,000 described species. Studies of gene function in squamates, however, have remained very limited, largely due to the lack of established genetic tools and suitable experimental systems. A major challenge for the development of CRISPR-based gene editing in these reptiles is that the isolation of fertilized oocytes or single-celled embryos is impractical for most species, given that fertilization occurs internally, the females of many species can store sperm, and simple methods for detecting ovulation are lacking. To overcome these challenges, we have developed a unique surgical approach in the brown anole lizard Anolis sagrei The procedure enables users to access and microinject unfertilized oocytes while they are still maturing within the lizard ovary. We describe here the methods to anesthetize adult female anoles, access the ovary through a surgical incision into the coelomic cavity, and microinject unfertilized oocytes with CRISPR-Cas9 ribonucleoprotein complexes to generate targeted mutations, enabling the routine production of gene-edited lizards.