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http://dx.doi.org/10.5713/ajas.19.0480

No excessive mutations in transcription activator-like effector nuclease-mediated α-1,3-galactosyltransferase knockout Yucatan miniature pigs  

Choi, Kimyung (Optipharm Inc.)
Shim, Joohyun (Optipharm Inc.)
Ko, Nayoung (Optipharm Inc.)
Park, Joonghoon (Department of International Agricultural Technology, Graduate School of International Agricultural Technology, Seoul National University)
Publication Information
Asian-Australasian Journal of Animal Sciences / v.33, no.2, 2020 , pp. 360-372 More about this Journal
Abstract
Objective: Specific genomic sites can be recognized and permanently modified by genome editing. The discovery of endonucleases has advanced genome editing in pigs, attenuating xenograft rejection and cross-species disease transmission. However, off-target mutagenesis caused by these nucleases is a major barrier to putative clinical applications. Furthermore, off-target mutagenesis by genome editing has not yet been addressed in pigs. Methods: Here, we generated genetically inheritable α-1,3-galactosyltransferase (GGTA1) knockout Yucatan miniature pigs by combining transcription activator-like effector nuclease (TALEN) and nuclear transfer. For precise estimation of genomic mutations induced by TALEN in GGTA1 knockout pigs, we obtained the whole-genome sequence of the donor cells for use as an internal control genome. Results: In-depth whole-genome sequencing analysis demonstrated that TALEN-mediated GGTA1 knockout pigs had a comparable mutation rate to homologous recombination-treated pigs and wild-type strain controls. RNA sequencing analysis associated with genomic mutations revealed that TALEN-induced off-target mutations had no discernable effect on RNA transcript abundance. Conclusion: Therefore, TALEN appears to be a precise and safe tool for generating genomeedited pigs, and the TALEN-mediated GGTA1 knockout Yucatan miniature pigs produced in this study can serve as a safe and effective organ and tissue resource for clinical applications.
Keywords
Transcription Activator-like Effector Nuclease (TALEN); Off-target; ${\alpha}$-1,3- Galactosyltransferase (GGTA1); Yucatan Miniature Pig; Whole-genome Sequencing; RNA Sequencing;
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