Generating Nonmosaic Mutants in Xenopus Using CRISPR-Cas in Oocytes

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• CRISPR-Cas9 genome editing often relies on nonhomologous end joining (NHEJ) for DNA repair, leading to random insertions/deletions (indels) and loss-of-function mutants.
• Homology-directed repair (HDR) offers precise DNA modification but occurs at lower frequencies in many systems.
• Xenopus oocytes exhibit higher HDR efficiency compared to eggs, presenting an opportunity for improved genome editing.

Purpose of the Study:

• To enhance the efficiency of precise genome editing in Xenopus using CRISPR-Cas9.
• To leverage the high HDR capacity of Xenopus oocytes for targeted DNA modifications.
• To develop a method for faster generation of homozygous mutants in Xenopus.

Main Methods:

• Utilized the oocyte host transfer technique for CRISPR-Cas9 component delivery into isolated Xenopus oocytes.
• Cultured injected oocytes in vitro for up to 5 days pre-fertilization to allow CRISPR-Cas9 activity and component degradation.
• Inhibited the NHEJ pathway using a DNA ligase IV inhibitor to further promote HDR.

Main Results:

• Successfully generated nonmosaic F0 heterozygous Xenopus with targeted indels.
• Achieved efficient, targeted insertion of small DNA fragments (73-104 nt) via HDR in Xenopus.
• Demonstrated germline transmission of mutations, enabling homozygous mutant generation in the F1 generation.

Conclusions:

• The developed method significantly improves the efficiency of precise genome editing in Xenopus.
• The use of oocyte host transfer and NHEJ inhibition accelerates the generation of homozygous Xenopus mutants.
• This approach provides a powerful tool for functional genomic studies in Xenopus.