Heritable CRISPR/Cas9-mediated targeted integration in Xenopus tropicalis

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• Xenopus tropicalis is an emerging vertebrate genetic model.
• A reliable gene knock-in method is currently lacking for this species.
• Precise genetic manipulation is crucial for advancing its utility in research.

Purpose of the Study:

• To establish a heritable gene knock-in method for Xenopus tropicalis.
• To enable precise genetic editing in this diploid frog model.
• To expand the applications of Xenopus tropicalis in studying human diseases.

Main Methods:

• Utilized the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system.
• Developed a concurrent cleavage strategy involving a modified donor vector with a Cas9/guide RNA cleavage site.
• Tested the method on three distinct genetic loci.

Main Results:

• Achieved efficient and heritable targeted integration at all three tested loci.
• Verified successful gene editing through germ-line transmission and Southern blot analyses.
• Demonstrated precise editing of coding sequences and expression of reporter genes, with no detectable off-target effects.

Conclusions:

• The developed CRISPR/Cas9-mediated concurrent cleavage strategy is effective for gene knock-in in Xenopus tropicalis.
• This method allows for precise genetic modifications, enhancing the frog's utility as a vertebrate model.
• The findings facilitate the creation of models for human inherited diseases using Xenopus tropicalis.