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Hum Mol Genet. 2017 Jul 15;26(14):2678-2689. doi: 10.1093/hmg/ddx154.

Quantitative assessment of timing, efficiency, specificity and genetic mosaicism of CRISPR/Cas9-mediated gene editing of hemoglobin beta gene in rhesus monkey embryos.

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Department of Animal Science, and Reproductive and Developmental Sciences Program, Michigan State University, East Lansing, MI 48824, USA.
California National Primate Research Center and Department of Obstetrics and Gynecology, University of California, Davis, Davis, CA 95616, USA.
Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA.
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.
Department of Pediatrics, Harvard Medical School and Harvard Stem Cell Institute, Harvard University, Boston, MA, USA.


Gene editing technologies offer new options for developing novel biomedical research models and for gene and stem cell based therapies. However, applications in many species demand high efficiencies, specificity, and a thorough understanding of likely editing outcomes. To date, overall efficiencies, rates of off-targeting and degree of genetic mosaicism have not been well-characterized for most species, limiting our ability to optimize methods. As a model gene for measuring these parameters of the CRISPR/Cas9 application in a primate species (rhesus monkey), we selected the β-hemoglobin gene (HBB), which also has high relevance to the potential application of gene editing and stem-cell technologies for treating human disease. Our data demonstrate an ability to achieve a high efficiency of gene editing in rhesus monkey zygotes, with no detected off-target effects at selected off-target loci. Considerable genetic mosaicism and variation in the fraction of embryonic cells bearing targeted alleles are observed, and the timing of editing events is revealed using a new model. The uses of Cas9-WT protein combined with optimized concentrations of sgRNAs are two likely areas for further refinement to enhance efficiency while limiting unfavorable outcomes that can be exceedingly costly for application of gene editing in primate species.

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