Production of light-coloured, low heat-absorbing Holstein Friesian cattle by precise embryo-mediated genome editing
Jingwei Wei![https://orcid.org/0000-0003-1048-2819](/media/client/orcid_16x16.png)
A
B
C
D
Abstract
Genome editing enables the introduction of beneficial sequence variants into the genomes of animals with high genetic merit in a single generation. This can be achieved by introducing variants into primary cells followed by producing a live animal from these cells by somatic cell nuclear transfer cloning. The latter step is associated with low efficiencies and developmental problems due to incorrect reprogramming of the donor cells, causing animal welfare concerns. Direct editing of fertilised one-cell embryos could circumvent this issue and might better integrate with genetic improvement strategies implemented by the industry.
In vitro fertilised zygotes were injected with TALEN editors and repair template to introduce a known coat colour dilution mutation in the PMEL gene. Embryo biopsies of injected embryos were screened by polymerase chain reaction and sequencing for intended biallelic edits before transferring verified embryos into recipients for development to term. Calves were genotyped and their coats scanned with visible and hyperspectral cameras to assess thermal energy absorption.
Multiple non-mosaic calves with precision edited genotypes were produced, including calves from high genetic merit parents. Compared to controls, the edited calves showed a strong coat colour dilution which was associated with lower thermal energy absorbance.
Although biopsy screening was not absolutely accurate, non-mosaic, precisely edited calves can be readily produced by embryo-mediated editing. The lighter coat colouring caused by the PMEL mutation can lower radiative heat gain which might help to reduce heat stress.
The study validates putative causative sequence variants to rapidly adapt grazing cattle to changing environmental conditions.
Keywords: cattle, coat color dilution, embryo-mediated, genome editing, heat stress, homology-directed repair, microinjection, P. Leu18del, PMEL, pre-melanosome protein 17, TALEN.
References
Akcakaya P, Bobbin ML, Guo JA, Malagon-Lopez J, Clement K, Garcia SP, Fellows MD, Porritt MJ, Firth MA, Carreras A, Baccega T, Seeliger F, Bjursell M, Tsai SQ, Nguyen NT, Nitsch R, Mayr LM, Pinello L, Bohlooly-Y M, Aryee MJ, Maresca M, Joung JK (2018) In vivo CRISPR editing with no detectable genome-wide off-target mutations. Nature 561(7723), 416-419.
| Crossref | Google Scholar | PubMed |
Andersson LS, Wilbe M, Viluma A, Cothran G, Ekesten B, Ewart S, Lindgren G (2013) Equine multiple congenital ocular anomalies and silver coat colour result from the pleiotropic effects of mutant PMEL. PLoS ONE 8(9), e75639.
| Crossref | Google Scholar | PubMed |
Becerril CM, Wilcox CJ, Lawlor TJ, Wiggans OR, Webb DW (1993) Effects of percentage of white coat color on holstein production and reproduction in a subtropical environment. Journal of Dairy Science 76(8), 2286-2291.
| Crossref | Google Scholar |
Bishop TF, Van Eenennaam AL (2020) Genome editing approaches to augment livestock breeding programs. Journal of Experimental Biology 223(Suppl_1), jeb207159.
| Crossref | Google Scholar |
Bols PEJ, Vandenheede JMM, Van Soom A, de Kruif A (1995) Transvaginal ovum pick-up (OPU) in the cow: a new disposable needle guidance system. Theriogenology 43(3), 677-687.
| Crossref | Google Scholar | PubMed |
Bonilla L, Block J, Denicol AC, Hansen PJ (2014) Consequences of transfer of an in vitro-produced embryo for the dam and resultant calf. Journal of Dairy Science 97(1), 229-239.
| Crossref | Google Scholar | PubMed |
Brinkman EK, Chen T, Amendola M, van Steensel B (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Research 42(22), e168.
| Crossref | Google Scholar | PubMed |
Brophy B, Smolenski G, Wheeler T, Wells D, L’Huillier P, Laible G (2003) Cloned transgenic cattle produce milk with higher levels of β-casein and κ-casein. Nature Biotechnology 21(2), 157-162.
| Crossref | Google Scholar | PubMed |
Carlson DF, Lancto CA, Zang B, Kim E-S, Walton M, Oldeschulte D, Seabury C, Sonstegard TS, Fahrenkrug SC (2016) Production of hornless dairy cattle from genome-edited cell lines. Nature Biotechnology 34(5), 479-481.
| Crossref | Google Scholar | PubMed |
Carvalho BP, Cunha ATM, Silva BDM, Sousa RV, Leme LO, Dode MAN, Melo EO (2019) Production of transgenic cattle by somatic cell nuclear transfer (SCNT) with the human granulocyte colony-stimulation factor (hG-CSF). Journal of Animal Science and Technology 61(2), 61-68.
| Crossref | Google Scholar | PubMed |
Cena K, Monteith JL (1975) Transfer processes in animal coats. I. Radiative transfer. Proceedings of the Royal Society B: Biological Sciences 188(1093), 377-393.
| Crossref | Google Scholar | PubMed |
Clark LA, Wahl JM, Rees CA, Murphy KE (2006) Retrotransposon insertion in SILV is responsible for merle patterning of the domestic dog. Proceedings of the National Academy of Sciences of the United States of America 103(5), 1376-1381.
| Crossref | Google Scholar | PubMed |
Davis SR, Spelman RJ, Littlejohn MD (2017) Breeding and Genetics Symposium:Breeding heat tolerant dairy cattle: the case for introgression of the “slick” prolactin receptor variant into Bos taurus dairy breeds. Journal of Animal Science 95(4), 1788-1800.
| Crossref | Google Scholar | PubMed |
Dreesen J, Drusedau M, Smeets H, de Die-Smulders C, Coonen E, Dumoulin J, Gielen M, Evers J, Herbergs J, Geraedts J (2008) Validation of preimplantation genetic diagnosis by PCR analysis: genotype comparison of the blastomere and corresponding embryo, implications for clinical practice. Molecular Human Reproduction 14(10), 573-579.
| Crossref | Google Scholar | PubMed |
Fujii T, Naito A, Hirayama H, Kashima M, Yoshino H, Hanamure T, Domon Y, Hayakawa H, Watanabe T, Moriyasu S, Kageyama S (2019) Potential of preimplantation genomic selection for carcass traits in Japanese Black cattle. Journal of Reproduction and Development 65(3), 251-258.
| Crossref | Google Scholar | PubMed |
Gebremedhin KG, Lee CN, Hillman PE, Brown-Brandl TM (2011) Body temperature and behavioral activities of four breeds of heifers in shade and full sun. Applied Engineering in Agriculture 27(6), 999-1006.
| Crossref | Google Scholar |
Georges M, Charlier C, Hayes B (2019) Harnessing genomic information for livestock improvement. Nature Reviews Genetics 20(3), 135-156.
| Crossref | Google Scholar | PubMed |
Hansen PJ (1990) Effects of coat colour on physiological responses to solar radiation in holsteins. Veterinary Record 127(13), 333-334.
| Google Scholar | PubMed |
Hellström AR, Watt B, Fard SS, Tenza D, Mannström P, Narfström K, Ekesten B, Ito S, Wakamatsu K, Larsson J, Ulfendahl M, Kullander K, Raposo G, Kerje S, Hallböök F, Marks MS, Andersson L (2011) Inactivation of Pmel alters melanosome shape but has only a subtle effect on visible pigmentation. PLoS Genetics 7(9), e1002285.
| Google Scholar | PubMed |
Hennig SL, Owen JR, Lin JC, Young AE, Ross PJ, Van Eenennaam AL, Murray JD (2020) Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10(1), 22309.
| Crossref | Google Scholar | PubMed |
Jivanji S, Harland C, Cole S, Brophy B, Garrick D, Snell R, Littlejohn M, Laible G (2021) The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis. BMC Genomics 22(1), 457.
| Crossref | Google Scholar | PubMed |
Kimura S, Hatakeyama T, Koutaka T, Kubo K, Morita S, Eguchi K, Saitoh K, Yamauchi K, Imai S, Kashimura A, Inenaga T, Matsumoto H (2022) PMEL p.Leu18del dilutes coat color of Kumamoto sub-breed of Japanese brown cattle. BMC Genomics 23(1), 694.
| Crossref | Google Scholar | PubMed |
Komáromy AM, Rowlan JS, La Croix NC, Mangan BG (2011) Equine multiple congenital ocular anomalies (MCOA) syndrome in PMEL17 (silver) mutant ponies: five cases. Veterinary Ophthalmology 14(5), 313-320.
| Crossref | Google Scholar | PubMed |
Lahola-Chomiak AA, Footz T, Nguyen-Phuoc K, Neil GJ, Fan B, Allen KF, Greenfield DS, Parrish RK, Linkroum K, Pasquale LR, Leonhardt RM, Ritch R, Javadiyan S, Craig JE, Allison WT, Lehmann OJ, Walter MA, Wiggs JL (2019) Non-synonymous variants in premelanosome protein (PMEL) cause ocular pigment dispersion and pigmentary glaucoma. Human Molecular Genetics 28(8), 1298-1311.
| Crossref | Google Scholar | PubMed |
Laible G, Cole S-A, Brophy B, Wei J, Leath S, Jivanji S, Littlejohn MD, Wells DN (2021) Holstein Friesian dairy cattle edited for diluted coat color as a potential adaptation to climate change. BMC Genomics 22(1), 856.
| Crossref | Google Scholar | PubMed |
Lawrenz B, El Khatib I, Liñán A, Bayram A, Arnanz A, Chopra R, De Munck N, Fatemi HM (2019) The clinicians’ dilemma with mosaicism-an insight from inner cell mass biopsies. Human Reproduction 34(6), 998-1010.
| Crossref | Google Scholar | PubMed |
Li C, Zhou S, Li Y, Li G, Ding Y, Li L, Liu J, Qu L, Sonstegard T, Huang X, Jiang Y, Chen Y, Petersen B, Wang X (2018) Trio-based deep sequencing reveals a low incidence of off-target mutations in the offspring of genetically edited goats. Frontiers in Genetics 9, 449.
| Crossref | Google Scholar | PubMed |
Liang D, Mikhalchenko A, Ma H, Marti Gutierrez N, Chen T, Lee Y, Park S-W, Tippner-Hedges R, Koski A, Darby H, Li Y, Van Dyken C, Zhao H, Wu K, Zhang J, Hou Z, So S, Han J, Park J, Kim C-J, Zong K, Gong J, Yuan Y, Gu Y, Shen Y, Olson SB, Yang H, Battaglia D, O’Leary T, Krieg SA, Lee DM, Wu DH, Duell PB, Kaul S, Kim J-S, Heitner SB, Kang E, Chen Z-J, Amato P, Mitalipov S (2023) Limitations of gene editing assessments in human preimplantation embryos. Nature Communications 14(1), 1219.
| Crossref | Google Scholar | PubMed |
Liu X, Wang Y, Guo W, Chang B, Liu J, Guo Z, Quan F, Zhang Y (2013) Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows. Nature Communications 4, 2565.
| Crossref | Google Scholar | PubMed |
Luo J, Song Z, Yu S, Cui D, Wang B, Ding F, Li S, Dai Y, Li N (2014) Efficient generation of myostatin (MSTN) biallelic mutations in cattle using zinc finger nucleases. PLoS ONE 9(4), e95225.
| Crossref | Google Scholar | PubMed |
Maia ASC, daSilva RG, Battiston Loureiro CM (2005a) Sensible and latent heat loss from the body surface of holstein cows in a tropical environment. International Journal of Biometeorology 50(1), 17-22.
| Crossref | Google Scholar | PubMed |
Maia ASC, Silva RGd, Bertipaglia ECA, Muñoz MC (2005b) Genetic variation of the hair coat properties and the milk yield of holstein cows managed under shade in a tropical environment. Brazilian Journal of Veterinary Research and Animal Science 42, 181-187.
| Crossref | Google Scholar |
McLean Z, Oback B, Laible G (2020) Embryo-mediated genome editing for accelerated genetic improvement of livestock. Frontiers of Agricultural Science and Engineering 7, 148-160.
| Crossref | Google Scholar |
Norris AL, Lee SS, Greenlees KJ, Tadesse DA, Miller MF, Lombardi HA (2020) Template plasmid integration in germline genome-edited cattle. Nature Biotechnology 38(2), 163-164.
| Crossref | Google Scholar | PubMed |
Park K-E, Frey JF, Waters J, Simpson SG, Coutu C, Plummer S, Campbell M, Donovan DM, Telugu BP (2020) One-step homology mediated CRISPR-Cas editing in zygotes for generating genome edited cattle. The CRISPR Journal 3(6), 523-534.
| Crossref | Google Scholar | PubMed |
Piyamongkol W, Bermudez MG, Harper JC, Wells D (2003) Detailed investigation of factors influencing amplification efficiency and allele drop-out in single cell PCR: implications for preimplantation genetic diagnosis. Molecular Human Reproduction 9(7), 411-420.
| Crossref | Google Scholar | PubMed |
Ponsart C, Le Bourhis D, Knijn H, Fritz S, Guyader-Joly C, Otter T, Lacaze S, Charreaux F, Schibler L, Dupassieux D, Mullaart E (2013) Reproductive technologies and genomic selection in dairy cattle. Reproduction, Fertility and Development 26(1), 12-21.
| Crossref | Google Scholar | PubMed |
Ruan J, Xu J, Chen-Tsai RY, Li K (2017) Genome editing in livestock: are we ready for a revolution in animal breeding industry? Transgenic Research 26(6), 715-726.
| Crossref | Google Scholar | PubMed |
Schmutz SM, Dreger DL (2013) Interaction of MC1R and PMEL alleles on solid coat colors in highland cattle. Animal Genetics 44(1), 9-13.
| Crossref | Google Scholar | PubMed |
Tutt DAR, Silvestri G, Serrano-Albal M, Simmons RJ, Kwong WY, Guven-Ates G, Canedo-Ribeiro C, Labrecque R, Blondin P, Handyside AH, Griffin DK, Sinclair KD (2021) Analysis of bovine blastocysts indicates ovarian stimulation does not induce chromosome errors, nor discordance between inner-cell mass and trophectoderm lineages. Theriogenology 161, 108-119.
| Crossref | Google Scholar | PubMed |
Varga L, Lénárt X, Zenke P, Orbán L, Hudák P, Ninausz N, Pelles Z, Szőke A (2020) Being merle: the molecular genetic background of the canine merle mutation. Genes 11(6), 660.
| Crossref | Google Scholar |
Vilarino M, Suchy FP, Rashid ST, Lindsay H, Reyes J, McNabb BR, van der Meulen T, Huising MO, Nakauchi H, Ross PJ (2018) Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep. Transgenic Research 27(6), 525-537.
| Crossref | Google Scholar | PubMed |
Wagner S, Cullum A, Wells DN, Laible G (2017) Taillessness in a cloned cow is not genetically transmitted. Cellular Reprogramming 19(6), 331-336.
| Crossref | Google Scholar | PubMed |
Wang X, Liu J, Niu Y, Li Y, Zhou S, Li C, Ma B, Kou Q, Petersen B, Sonstegard T, Huang X, Jiang Y, Chen Y (2018) Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep. BMC Genomics 19(1), 397.
| Crossref | Google Scholar | PubMed |
Wei J, Wagner S, Lu D, Maclean P, Carlson DF, Fahrenkrug SC, Laible G (2015) Efficient introgression of allelic variants by embryo-mediated editing of the bovine genome. Scientific Reports 5, 11735.
| Crossref | Google Scholar | PubMed |
Wei J, Wagner S, Maclean P, Brophy B, Cole S, Smolenski G, Carlson DF, Fahrenkrug SC, Wells DN, Laible G (2018) Cattle with a precise, zygote-mediated deletion safely eliminate the major milk allergen beta-lactoglobulin. Scientific Reports 8(1), 7661.
| Crossref | Google Scholar | PubMed |
Wei J, Brophy B, Cole S-A, Moormann J, Boch J, Laible G (2022) Cytoplasmic injection of zygotes to genome edit naturally occurring sequence variants into bovine embryos. Frontiers in Genetics 13, 925913.
| Crossref | Google Scholar | PubMed |
Wells DN (2005) Animal cloning: problems and prospects. Revue Scientifique et Technique 24(1), 251-264.
| Crossref | Google Scholar | PubMed |
Wu H, Wang Y, Zhang Y, Yang M, Lv J, Liu J, Zhang Y (2015) TALE nickase-mediated SP110 knockin endows cattle with increased resistance to tuberculosis. Proceedings of the National Academy of Sciences of the United States of America 112(13), E1530-E1539.
| Crossref | Google Scholar | PubMed |
Yee J-K (2016) Off-target effects of engineered nucleases. The FEBS Journal 283(17), 3239-3248.
| Crossref | Google Scholar | PubMed |
Young AE, Mansour TA, McNabb BR, Owen JR, Trott JF, Brown CT, Van Eenennaam AL (2020) Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull. Nature Biotechnology 38(2), 225-232.
| Crossref | Google Scholar | PubMed |
Zhang J, Liu J, Yang W, Cui M, Dai B, Dong Y, Yang J, Zhang X, Liu D, Liang H, Cang M (2019) Comparison of gene editing efficiencies of CRISPR/Cas9 and TALEN for generation of MSTN knock-out cashmere goats. Theriogenology 132, 1-11.
| Crossref | Google Scholar | PubMed |