Abstract
Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency for the introduction of large DNA sequences in zygotes is low. The homology-mediated end joining (HMEJ) approach has been shown to improve knock-in efficiency in non-dividing cells and to harness HDR after direct injection of embryos. The knock-in efficiency for a 1.8 kb gene was contrasted when combining microinjection of a gRNA/Cas9 ribonucleoprotein complex with a traditional HR donor template or an HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach will facilitate the one-step introduction of gene constructs at a specific location of the bovine genome and contribute to the next generation of elite cattle.
Highlights
Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging
The predominant method for creating a gene insertion or knock-in (KI) using genome editing uses a two-step process, in which first a primary cell line is modified in culture and used for somatic cell nuclear transfer (SCNT) cloning to create an animal with the intended gene modification[5]
There have been no successful KIs of large segments of DNA using cytoplasmic injection (CPI) of one-cell embryos and only two reports showing the successful introduction of single nucleotide polymorphisms (SNPs) or precise deletions in bovine embryos[9,10]
Summary
Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Genome editing offers an opportunity to augment livestock breeding programs by directly introducing targeted genetic variation to improve desired traits such as disease resistance and resilience[2,3,4]. The predominant method for creating a gene insertion or knock-in (KI) using genome editing uses a two-step process, in which first a primary cell line is modified in culture and used for somatic cell nuclear transfer (SCNT) cloning to create an animal with the intended gene modification[5]. Many attempts have been made to increase the rate of homologous recombination (HR), or decrease the rate of non-homologous end joining (NHEJ) for gene insertion, when using the CRISPR/Cas[9] system via CPI of zygotes[13] These approaches have been unsuccessful in bovine embryos as HR is primarily restricted to actively dividing c ells[14]. The primary repair mechanism for DNA double-strand breaks
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