Efficient introgression of allelic variants by embryo-mediated editing of the bovine genome

Abstract

The recent development of designer nucleases allows for the efficient and precise introduction of genetic change into livestock genomes. Most studies so far have focused on the introduction of random mutations in cultured cells and the use of nuclear transfer to generate animals with edited genotypes. To circumvent the intrinsic uncertainties of random mutations and the inefficiencies of nuclear transfer we directed our efforts to the introduction of specific genetic changes by homology-driven repair directly in in vitro produced embryos. Initially, we injected zinc finger nuclease (ZFN)-encoding mRNA or DNA into bovine zygotes to verify cleavage activity at their target site within the gene for beta-lactoglobulin (LGB) and detected ZFN-induced random mutations in 30% to 80% of embryos. Next, to precisely change the LGB sequence, we co-injected ZFNs or transcription activator-like effector nucleases (TALENs) with DNA oligonucleotides (ODNs). Analysis of co-injected embryos showed targeted changes in up to 33% (ZFNs) and 46% (TALENs) of blastocysts. Deep sequence analysis of selected embryos revealed contributions of the targeted LGB allele can reach 100% which implies that genome editing by zygote injections can facilitate the one-step generation of non-mosaic livestock animals with pre-designed biallelic modifications.

Evaluation of Mosaicism and Off Target Mutations in CRISPR-Mediated Genome Edited Bovine Embryos

“…Studies utilizing transcription activator-like effector nucleases (TALENs) have demonstrated lower mosaicism rates than we observed here; however, the proportion of edited embryos tends to be lower as well 6,7 . A study employing a zinc finger nuclease (ZFN) in bovine embryos demonstrated both high embryo editing efficiency and mosaicism rates as compared to those found in TALEN edited embryos 8 . However, the prevalence of mosaicism was reduced when injecting embryos at 8hpi compared to 18hpi, before S-phase had occurred 8 .…”

“…A study employing a zinc finger nuclease (ZFN) in bovine embryos demonstrated both high embryo editing efficiency and mosaicism rates as compared to those found in TALEN edited embryos 8 . However, the prevalence of mosaicism was reduced when injecting embryos at 8hpi compared to 18hpi, before S-phase had occurred 8 . While we were able to induce mutations in embryos at a high rate, we also observed a high level of mosaicism when injecting 18hpi.…”

Embryo-mediated genome editing for accelerated genetic improvement of livestock

“…By contrast, editing during S phase occurs while each chromosomal DNA strand is being replicated into two sister chromatids, doubling the number of target sites, but coinciding with the DSB repair machinery being most active [38] . Previous work by our group showed that there was no significant difference in the proportion of HDR-edited embryos between delivering the nuclease and repair template at G 1 (8 h) vs. S phase (18 h) [27] . These experiments were mostly performed with ZFNs delivered as a plasmid, with the DNA cleavage expected to be faster if the nuclease was delivered as a protein.…”

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

“…These results are similar to efficiencies previously reported in livestock species for KI via cytoplasmic injection of an HR template (5.7% in sheep embryos 16 and 6.5% in pig embryos 17 ). While there have been reports of using genome editors and donor template ODNs to induce single nucleotide polymorphisms or precise deletions of bovine embryos 18,19 , this is the first reported use of the CRISPR/Cas9 system for efficient KI of a large DNA segment (1.8kb).…”