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Genome Biol. 2019 Jan 17;20(1):14. doi: 10.1186/s13059-019-1625-3.

iGUIDE: an improved pipeline for analyzing CRISPR cleavage specificity.

Nobles CL1, Reddy S1, Salas-McKee J2,3,4,5, Liu X2,3,4,5, June CH2,3,4,5, Melenhorst JJ2,3,4,5, Davis MM2,3,4,5, Zhao Y2,3,4,5, Bushman FD6.

Author information

1
Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 3610 Hamilton Walk, Philadelphia, PA, 19104-6076, USA.
2
Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
3
Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA.
4
Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA.
5
Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, USA.
6
Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 3610 Hamilton Walk, Philadelphia, PA, 19104-6076, USA. bushman@mail.med.upenn.edu.

Abstract

Genome engineering methods have advanced greatly with the development of programmable nucleases, but methods for quantifying on- and off-target cleavage sites and associated deletions remain nascent. Here, we report an improvement of the GUIDE-seq method, iGUIDE, which allows filtering of mispriming events to clarify the true cleavage signal. Using iGUIDE, we specify the locations of Cas9-guided cleavage for four guide RNAs, characterize associated deletions, and show that naturally occurring background DNA double-strand breaks are associated with open chromatin, gene dense regions, and chromosomal fragile sites. iGUIDE is available from https://github.com/cnobles/iGUIDE .

PMID:
30654827
PMCID:
PMC6337799
DOI:
10.1186/s13059-019-1625-3
[Indexed for MEDLINE]
Free PMC Article

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