Enhancing the genome editing toolbox: genome wide CRISPR arrayed libraries

CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome w...

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Published inScientific reports Vol. 7; no. 1; pp. 2244 - 9
Main Authors Metzakopian, Emmanouil, Strong, Alex, Iyer, Vivek, Hodgkins, Alex, Tzelepis, Konstantinos, Antunes, Liliana, Friedrich, Mathias J, Kang, Qiaohua, Davidson, Teresa, Lamberth, Jacob, Hoffmann, Christina, Davis, Gregory D, Vassiliou, George S, Skarnes, William C, Bradley, Allan
Format Journal Article
LanguageEnglish
Published England Nature Publishing Group 22.05.2017
Nature Publishing Group UK
Nature Portfolio
Subjects
Animals
Cell lines
Cloning
CRISPR
CRISPR-Cas Systems
Deoxyribonucleic acid
DNA
Gene Editing - methods
Gene Library
Genes
Genetic Vectors
Genome editing
Genome-Wide Association Study - methods
Genomes
GPI-Linked Proteins - metabolism
Humans
Labeling
Lentivirus - genetics
Mice
Phenotype
RNA, Guide, CRISPR-Cas Systems
Signal Transduction
Transposase
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Summary:CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.
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ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-017-01766-5