Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing

Area of Science:

• Genetics
• Animal Models
• Biotechnology

Background:

• Genome editing in pigs is valuable but challenging.
• Existing methods are time-consuming and expensive.
• Advanced tools are needed to facilitate gene editing in pigs.

Purpose of the Study:

• To establish a pig line with Cre-inducible Cas9 expression for versatile genome editing.
• To demonstrate the utility of this pig line for both ex vivo and in vivo genetic modifications.
• To showcase its application in complex genetic manipulations for disease modeling.

Main Methods:

• Generation of a pig line expressing Cre-inducible Cas9.
• Ex vivo genome editing in fibroblast cells (single/multi-gene edits, chromosome rearrangements).
• In vivo genetic modifications using Cre recombinase and single-guide RNAs (sgRNAs).

Main Results:

• Successful establishment of a Cre-inducible Cas9 pig line.
• Demonstrated efficient ex vivo editing, including complex modifications.
• Achieved simultaneous inactivation of five tumor suppressor genes (TP53, PTEN, APC, BRCA1, BRCA2) and activation of an oncogene (KRAS) in vivo.
• Observed rapid lung tumor development as a proof of principle.

Conclusions:

• The developed pig line is a powerful tool for temporal gene function dissection in vivo.
• This model streamlines the production of genome-edited pigs for disease modeling.
• Facilitates agricultural, biomedical, and pharmaceutical applications through efficient genome editing.