Cytoplasmic injection of zygotes to genome edit naturally occurring sequence variants into bovine embryos

authored by

Jingwei Wei, Brigid Brophy, Sally Ann Cole, Jannis Moormann, Jens Boch, Gӧtz Laible

Abstract

Genome editing provides opportunities to improve current cattle breeding strategies through targeted introduction of natural sequence variants, accelerating genetic gain. This can be achieved by harnessing homology-directed repair mechanisms following editor-induced cleavage of the genome in the presence of a repair template. Introducing the genome editors into zygotes and editing in embryos has the advantage of uncompromised development into live animals and alignment with contemporary embryo-based improvement practices. In our study, we investigated the potential to introduce sequence variants, known from the pre-melanosomal protein 17 (PMEL) and prolactin receptor (PRLR) genes, and produce non-mosaic, edited embryos, completely converted into the precision genotype. Injection of gRNA/Cas9 editors into bovine zygotes to introduce a 3 bp deletion variant into the PMEL gene produced up to 11% fully converted embryos. The conversion rate was increased to up to 48% with the use of TALEN but only when delivered by plasmid. Testing three gRNA/Cas9 editors in the context of several known PRLR sequence variants, different repair template designs and delivery as DNA, RNA or ribonucleoprotein achieved full conversion rates up to 8%. Furthermore, we developed a biopsy-based screening strategy for non-mosaic embryos which has the potential for exclusively producing non-mosaic animals with intended precision edits.

Organisation(s)

Section Plant Molecular Biology and Plant Proteomics
Section Plant Biotechnology

External Organisation(s)

AgResearch
University of Auckland

Type

Article

Journal

Frontiers in Genetics

Volume

13

ISSN

1664-8021

Publication date

11.07.2022

Publication status

Published

Peer reviewed

Yes

ASJC Scopus subject areas

Molecular Medicine, Genetics, Genetics(clinical)

Electronic version(s)

https://doi.org/10.3389/fgene.2022.925913 (Access: Open)