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J Genet Genomics. 2015 Jun 20;42(6):301-9. doi: 10.1016/j.jgg.2015.03.011. Epub 2015 Apr 18.

A genome-wide CRISPR library for high-throughput genetic screening in Drosophila cells.

Author information

1
MRC Functional Genomics Unit, University of Oxford, Department of Physiology, Anatomy and Genetics, South Parks Road, Oxford, OX1 3PT, United Kingdom; Genome Engineering Oxford, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, United Kingdom. Electronic address: andrew.bassett@path.ox.ac.uk.
2
MRC Functional Genomics Unit, University of Oxford, Department of Physiology, Anatomy and Genetics, South Parks Road, Oxford, OX1 3PT, United Kingdom.
3
MRC Functional Genomics Unit, University of Oxford, Department of Physiology, Anatomy and Genetics, South Parks Road, Oxford, OX1 3PT, United Kingdom. Electronic address: jilong.liu@dpag.ox.ac.uk.

Abstract

The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines, enabling screening for cellular phenotypes resulting from genetic aberrations. Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi. This is in part due to the lower degree of redundancy between genes in this organism, whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes. The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques, but allows analysis over longer periods of time which can be critical for certain phenotypes. In this study, we have designed and built a genome-wide CRISPR library covering 13,501 genes, among which 8989 genes are targeted by three or more independent single guide RNAs (sgRNAs). Moreover, we describe strategies to monitor the population of guide RNAs by high throughput sequencing (HTS). We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes, and as a source of guide RNA designs for future studies.

KEYWORDS:

CRISPR/Cas9; Drosophila; Genome-wide library

PMID:
26165496
PMCID:
PMC4508376
DOI:
10.1016/j.jgg.2015.03.011
[Indexed for MEDLINE]
Free PMC Article

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