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Sci Rep. 2019 Feb 7;9(1):1662. doi: 10.1038/s41598-018-33533-5.

Simultaneous targeting of linked loci in mouse embryos using base editing.

Author information

1
Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland, 20892, USA. hyekyung.lee@nih.gov.
2
Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland, 20892, USA.
3
Genomics Core, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland, 20892, USA.
4
Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, 02142, USA.
5
Howard Hughes Medical Institute, Harvard University, Cambridge, MA, 02138, USA.
6
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, 02138, USA.
7
Transgenic Core, National Heart, Lung, and Blood Institute, US National Institutes of Health, Bethesda, Maryland, 20892, USA.
8
Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland, 20892, USA. lotharh@niddk.nih.gov.

Abstract

A particular challenge in genome engineering has been the simultaneous introduction of mutations into linked (located on the same chromosome) loci. Although CRISPR/Cas9 has been widely used to mutate individual sites, its application in simultaneously targeting of linked loci is limited as multiple nearby double-stranded DNA breaks created by Cas9 routinely result in the deletion of sequences between the cleavage sites. Base editing is a newer form of genome editing that directly converts C∙G-to-T∙A, or A∙T-to-G∙C, base pairs without introducing double-stranded breaks, thus opening the possibility to generate linked mutations without disrupting the entire locus. Through the co-injection of two base editors and two sgRNAs into mouse zygotes, we introduced C∙G-to-T∙A transitions into two cytokine-sensing transcription factor binding sites separated by 9 kb. We determined that one enhancer activates the two flanking genes in mammary tissue during pregnancy and lactation. The ability to introduce linked mutations simultaneously in one step into the mammalian germline has implications for a wide range of applications, including the functional analysis of linked cis-elements creating disease models and correcting pathogenic mutations.

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