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Nat Commun. 2017 May 31;8:15464. doi: 10.1038/ncomms15464.

CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome.

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.
2
Department of Biomedical Science and Engineering, Konkuk University, Seoul 05029, Republic of Korea.
3
Department of Cell and Developmental Biology &Dental Research Institute, Seoul National University, Seoul 110-749, Republic of Korea.
4
Department of Life Systems, Sookmyung Women's University, Seoul 140-742, Republic of Korea.
5
Transgenic Core, National Heart, Lung, and Blood Institute, US National Institutes of Health, Bethesda, Maryland 20892, USA.

Abstract

Although CRISPR/Cas9 genome editing has provided numerous opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome. Here we interrogate the molecular consequences of CRISPR/Cas9-mediated deletions at 17 sites in four loci of the mouse genome. We sequence targeted sites in 632 founder mice and analyse 54 established lines. While the median deletion size using single sgRNAs is 9 bp, we also obtain large deletions of up to 600 bp. Furthermore, we show unreported asymmetric deletions and large insertions of middle repetitive sequences. Simultaneous targeting of distant loci results in the removal of the intervening sequences. Reliable deletion of juxtaposed sites is only achieved through two-step targeting. Our findings also demonstrate that an extended analysis of F1 genotypes is required to obtain conclusive information on the exact molecular consequences of targeting events.

PMID:
28561021
PMCID:
PMC5460021
DOI:
10.1038/ncomms15464
[Indexed for MEDLINE]
Free PMC Article

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