期刊
SCIENTIFIC REPORTS
卷 10, 期 1, 页码 -出版社
NATURE RESEARCH
DOI: 10.1038/s41598-020-78264-8
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资金
- NIH Shared Instrumentation Grant [1S10OD010786-01]
- Biotechnology Risk Assessment Grant Program competitive Grant from the U.S. Department of Agriculture [2015-33522-24106]
- Academic Federation Innovation Development Award at UC Davis
- Russell L. Rustici Rangeland and Cattle Research Endowment in the College of Agricultural and Environmental Science at UC Davis
- California Agricultural Experiment Station of the University of California, Davis
- Henry A. Jastro Research Fellowship in the College of Agricultural and Environmental Science at UC Davis
- National Institute for Food and Agriculture National Needs Graduate and Postgraduate Fellowship from the U.S. Department of Agriculture [2017-38420-26790]
- NIFA [2015-33522-24106, 810183] Funding Source: Federal RePORTER
The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrated that the use of gRNA/Cas9 ribonucleoprotein complex resulted in a high editing efficiency at three different loci in bovine embryos and decreased levels of mosaicism relative to Cas9 mRNA. Additional optimization will be required to further reduce mosaicism to levels that make single-step embryo editing in cattle commercially feasible.
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