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

Gene edited pigs have extensive and important application value in the fields of agriculture and biomedicine. With the increasing demand in medical research and agricultural markets, more and more application scenarios require gene edited pigs to possess two or even more advantageous phenotypes simultaneously. The current production of multi gene edited pigs is inefficient, time-consuming, and costly, and there is an urgent need to develop efficient and accurate multi gene editing application technologies. The polycistronic tRNA-gRNA-processing system (PTG), developed based on endogenous tRNA self-processing systems, has been shown to exhibit efficient multi gene editing in plants. This study aims to combine a PTG strategy with multiple gRNA production functions with an adenine base editor (ABE) to test its feasibility for efficient and precise multi gene base editing in pig cells. The results indicate that the PTG based integrated ABE plasmid can perform efficient base editing at multiple gene loci in pig cells. And while the gene editing efficiency was significantly improved, no indel and sgRNA dependent off target effects caused by DSB were detected. This work permit will provide a solid foundation for the production of multi gene edited pigs with agricultural and medical applications.