TY - JOUR

T1 - Cytoplasmic injection of zygotes to genome edit naturally occurring sequence variants into bovine embryos

AU - Wei, Jingwei

AU - Brophy, Brigid

AU - Cole, Sally Ann

AU - Moormann, Jannis

AU - Boch, Jens

AU - Laible, Gӧtz

N1 - Funding Information: JW, BB, S-AC and GL are employees of AgResearch and received co-funding from CRV Ltd. and Livestock Improvement Corporation in support of the MBIE research grant. JB is a part owner of patents concerning the use of TALEs and TALENs. All authors declare no other competing interests. Funding Information: This work was funded through the Ministry of Business, Innovation and Employment (MBIE) ( https://www.mbie.govt.nz/ ) Endeavour Funds CONT-62639-ENDRP-AGR, CRV Ltd. and Livestock Improvement Corporation. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

PY - 2022/7/11

Y1 - 2022/7/11

N2 - 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.

AB - 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.

KW - Cas9

KW - cattle

KW - genome editing

KW - homology-directed repair

KW - PMEL

KW - PRLR

KW - slick

KW - TALEN

UR - http://www.scopus.com/inward/record.url?scp=85134700961&partnerID=8YFLogxK

U2 - 10.3389/fgene.2022.925913

DO - 10.3389/fgene.2022.925913

M3 - Article

AN - SCOPUS:85134700961

VL - 13

JO - Frontiers in Genetics

JF - Frontiers in Genetics

SN - 1664-8021

M1 - 925913

ER -