Application of Multiple Base-Editing Mediated by Polycistronic tRNA-gRNA-Processing System in Pig Cells

Area of Science:

• Agricultural Science
• Biomedical Science
• Gene Editing Technologies

Background:

• Gene-edited pigs are vital for agriculture and biomedicine, with increasing demand for animals possessing multiple advantageous traits.
• Current methods for creating multi-gene edited pigs are inefficient, costly, and time-consuming, necessitating advanced technologies.

Purpose of the Study:

• To evaluate the feasibility of a polycistronic tRNA-gRNA-processing system (PTG) combined with an adenine base editor (ABE) for efficient multi-gene base editing in pig cells.
• To establish a foundation for producing pigs with multiple desired agricultural and medical applications.

Main Methods:

• Integration of a PTG strategy for multiple guide RNA production with an adenine base editor (ABE).
• Application of the combined system in pig cells to assess multi-gene base editing efficiency and accuracy.

Main Results:

• The PTG-based integrated ABE plasmid demonstrated efficient base editing across multiple gene loci in pig cells.
• Significantly improved gene editing efficiency was observed.
• No detectable indels or double-strand break (DSB)-dependent off-target mutations were found.

Conclusions:

• The PTG strategy combined with ABE provides an efficient and precise method for multi-gene base editing in pigs.
• This technology offers a robust foundation for developing pigs with enhanced agricultural and biomedical traits.