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

Area of Science:

• Herpetology
• Developmental Biology
• Molecular Genetics

Background:

• Squamates (lizards and snakes) are highly diverse reptiles, but studies of gene function are limited due to a lack of genetic tools.
• Existing gene-editing approaches like CRISPR-Cas9 are challenging in squamates because isolating early-stage embryos or fertilized oocytes is impractical.

Purpose of the Study:

• To develop a novel method for gene editing in squamates, specifically targeting the brown anole lizard (Anolis sagrei).
• To overcome the technical hurdles associated with internal fertilization and sperm storage in reptiles for genetic manipulation.

Main Methods:

• A surgical procedure was developed to access the ovary in adult female brown anole lizards.
• Unfertilized oocytes were microinjected with CRISPR-Cas9 ribonucleoprotein complexes while still maturing within the ovary.
• Methods included anesthesia, coelomic cavity incision, and targeted oocyte microinjection.

Main Results:

• The developed surgical approach enables direct access to and microinjection of immature oocytes in vivo.
• This method facilitates the routine generation of gene-edited lizards with targeted mutations.
• Successful application of CRISPR-Cas9 for generating genetically modified anoles was demonstrated.

Conclusions:

• This innovative technique overcomes significant barriers to squamate gene editing, particularly the difficulty in obtaining early embryos.
• The described method provides a viable pathway for advancing functional genomic studies in lizards and potentially other reptiles.
• This breakthrough opens new avenues for research in reptile development, evolution, and conservation genetics.