In vitro gene editing using primary cells derived from Cas9-expressing pigs

Area of Science:

• Genetics
• Animal Biotechnology
• Molecular Biology

Background:

• Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology enables gene editing in pigs.
• Generating genetically engineered pigs is currently time- and labor-intensive.
• Existing in vivo gene-editing methods face efficiency challenges due to the large size of the Cas9 nuclease.

Purpose of the Study:

• To develop a more efficient platform for generating gene-edited pigs.
• To create a pig model with constitutive Cas9 expression for simplified gene editing.
• To establish a resource for both in vivo and in vitro gene editing applications.

Main Methods:

• Generated a transgenic pig by integrating the Cas9 gene into the ROSA26 locus.
• Confirmed constitutive Cas9 expression in various tissues of the generated pig.
• Assessed the fertility of the Cas9-expressing pig.
• Performed in vitro gene editing experiments using primary cells from the pig.

Main Results:

• Successfully generated a fertile pig with constitutive Cas9 expression.
• Demonstrated effective gene deletion in vitro by adding only single guide RNAs (sgRNAs) to primary cells.
• The ROSA26 locus facilitated stable and widespread Cas9 expression.

Conclusions:

• The developed Cas9-expressing pig serves as an effective platform for gene editing.
• This model significantly simplifies and accelerates the generation of genetically engineered pigs.
• The platform holds potential for advancing agricultural and biomedical research through efficient gene editing.