In:
The FEBS Journal, Wiley, Vol. 286, No. 23 ( 2019-12), p. 4675-4692
Abstract:
The ability to alter single bases without homology directed repair ( HDR ) of double‐strand breaks provides a potential solution for editing livestock genomes for economic traits, which are often multigenic. Progress toward multiplex editing in large animals has been hampered by the costly inefficiencies of HDR via microinjection of in vitro manipulated embryos. Here, we designed sg RNA s to induce nonsense codons (C‐to‐T transitions) at four target sites in caprine FGF 5 , which is a crucial regulator of hair length in mammals. Initial transfections of the third generation Base Editor ( BE 3) plasmid and four different sg RNA s into caprine fibroblasts were ineffective in altering FGF 5 . In contrast, all five progenies produced from microinjected single‐cell embryos had alleles with a targeted nonsense mutation. The effectiveness of BE 3 to make single base changes varied considerably based on sg RNA design. In addition, the rate of mosaicism differed between animals, target sites, and tissue type. The phenotypic effects on hair fiber were characterized by hematoxylin and eosin, immunofluorescence staining, and western blotting. Differences in morphology were detectable, even though mosaicism was probably affecting the levels of FGF 5 expression. PCR amplicon and whole‐genome resequencing analyses for off‐target changes caused by BE 3 were low at a genome‐wide scale. This study provided the first evidence of base editing in large mammals produced from microinjected single‐cell embryos. Our results support further optimization of BE s for introgressing complex human disease alleles into large animal models, to evaluate potential genetic improvement of complex health and production traits in a single generation.
Type of Medium:
Online Resource
ISSN:
1742-464X
,
1742-4658
DOI:
10.1111/febs.v286.23
Language:
English
Publisher:
Wiley
Publication Date:
2019
detail.hit.zdb_id:
2172518-4
SSG:
12
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