iGUIDE: an improved pipeline for analyzing CRISPR cleavage specificity

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...

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Published inGenome Biology Vol. 20; no. 1; p. 14
Main Authors Nobles, Christopher L, Reddy, Shantan, Salas-McKee, January, Liu, Xiaojun, June, Carl H, Melenhorst, J Joseph, Davis, Megan M, Zhao, Yangbing, Bushman, Frederic D
Format Journal Article
LanguageEnglish
Published England BioMed Central 17.01.2019
BMC
Subjects
Binding sites
Chromatin
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR
Deoxyribonucleic acid
Design
DNA
DNA damage
Fragile sites
Genetic Engineering - methods
Genome editing
Genomes
Immunotherapy
Lymphocytes
Methods
Nuclease
Short Report
Software
Stem cells
T cell receptors
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Summary: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 .
Bibliography:ObjectType-Article-2
SourceType-Scholarly Journals-1
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ISSN:1474-760X
1474-7596
1474-760X
DOI:10.1186/s13059-019-1625-3