Production of MSTN knockout porcine cells using adenine base-editing-mediated exon skipping.

Autor: Yang SP; Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Sciences and Engineering, Foshan University, Foshan, 528225, China., Zhu XX; Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Sciences and Engineering, Foshan University, Foshan, 528225, China. zhu_xiangxing@126.com.; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China. zhu_xiangxing@126.com., Qu ZX; Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Sciences and Engineering, Foshan University, Foshan, 528225, China., Chen CY; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Wu YB; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Wu Y; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Luo ZD; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Wang XY; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., He CY; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Fang JW; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Wang LQ; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Hong GL; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Zheng ST; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Zeng JM; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Yan AF; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Feng J; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Liu L; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Zhang XL; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Zhang LG; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China., Miao K; Centre for Precision Medicine Research and Training, Faculty of Health Sciences, University of Macau, Macau SAR, China. kaimiao@um.edu.mo., Tang DS; Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Sciences and Engineering, Foshan University, Foshan, 528225, China. tangdsh@163.com.; Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China. tangdsh@163.com.
Jazyk: angličtina
Zdroj: In vitro cellular & developmental biology. Animal [In Vitro Cell Dev Biol Anim] 2023 Apr; Vol. 59 (4), pp. 241-255. Date of Electronic Publication: 2023 Apr 26.
DOI: 10.1007/s11626-023-00763-5
Abstrakt: Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9 and cytosine base editing (CBE) technologies, adenine base editing (ABE) shows better safety and accuracy in gene modification. However, because of the characteristics of gene sequences, the ABE system cannot be widely used in gene knockout. Alternative splicing of mRNA is an important biological mechanism in eukaryotes for the formation of proteins with different functional activities. The splicing apparatus recognizes conserved sequences of the 5' end splice donor and 3' end splice acceptor motifs of introns in pre-mRNA that can trigger exon skipping, leading to the production of new functional proteins, or causing gene inactivation through frameshift mutations. This study aimed to construct a MSTN knockout pig by inducing exon skipping with the aid of the ABE system to expand the application of the ABE system for the preparation of knockout pigs. In this study, first, we constructed ABEmaxAW and ABE8eV106W plasmid vectors and found that their editing efficiencies at the targets were at least sixfold and even 260-fold higher than that of ABEmaxAW by contrasting the editing efficiencies at the gene targets of endogenous CD163, IGF2, and MSTN in pigs. Subsequently, we used the ABE8eV106W system to realize adenine base (the base of the antisense strand is thymine) editing of the conserved splice donor sequence (5'-GT) of intron 2 of the porcine MSTN gene. A porcine single-cell clone carrying a homozygous mutation (5'-GC) in the conserved sequence (5'-GT) of the intron 2 splice donor of the MSTN gene was successfully generated after drug selection. Unfortunately, the MSTN gene was not expressed and, therefore, could not be characterized at this level. No detectable genomic off-target edits were identified by Sanger sequencing. In this study, we verified that the ABE8eV106W vector had higher editing efficiency and could expand the editing scope of ABE. Additionally, we successfully achieved the precise modification of the alternative splice acceptor of intron 2 of the porcine MSTN gene, which may provide a new strategy for gene knockout in pigs.
(© 2023. The Society for In Vitro Biology.)
Databáze: MEDLINE
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