Details
| Original language | English |
|---|---|
| Article number | 925913 |
| Journal | Frontiers in Genetics |
| Volume | 13 |
| Publication status | Published - 11 Jul 2022 |
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.
Keywords
- Cas9, cattle, genome editing, homology-directed repair, PMEL, PRLR, slick, TALEN
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Medicine
- Biochemistry, Genetics and Molecular Biology(all)
- Genetics
- Medicine(all)
- Genetics(clinical)
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In: Frontiers in Genetics, Vol. 13, 925913, 11.07.2022.
Research output: Contribution to journal › Article › Research › peer review
}
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 -