Dataset Information

A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing.

ABSTRACT: BACKGROUND: CRISPR-Cas9 is a revolutionary genome editing technique that allows for efficient and directed alterations of the eukaryotic genome. This relatively new technology has already been used in a large number of 'loss of function' experiments in cultured cells. Despite its simplicity and efficiency, screening for mutated clones remains time-consuming, laborious and/or expensive. RESULTS: Here we report a high-throughput screening strategy that allows parallel screening of up to 96 clones, using next-generation sequencing. As a proof of principle, we used CRISPR-Cas9 to disrupt the coding sequence of the homeobox gene, Evx1 in mouse embryonic stem cells. We screened 67 CRISPR-Cas9 transfected clones simultaneously by next-generation sequencing on the Ion Torrent PGM. We were able to identify both homozygous and heterozygous Evx1 mutants, as well as mixed clones, which must be identified to maintain the integrity of subsequent experiments. CONCLUSIONS: Our CRISPR-Cas9 screening strategy could be widely applied to screen for CRISPR-Cas9 mutants in a variety of contexts including the generation of mutant cell lines for in vitro research, the generation of transgenic organisms and for assessing the veracity of CRISPR-Cas9 homology directed repair. This technique is cost and time-effective, provides information on clonal heterogeneity and is adaptable for use on various sequencing platforms.

SUBMITTER: Bell CC

PROVIDER: S-EPMC4246457 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing.

Bell Charles C CC Magor Graham W GW Gillinder Kevin R KR Perkins Andrew C AC

BMC genomics 20141120

<h4>Background</h4>CRISPR-Cas9 is a revolutionary genome editing technique that allows for efficient and directed alterations of the eukaryotic genome. This relatively new technology has already been used in a large number of 'loss of function' experiments in cultured cells. Despite its simplicity and efficiency, screening for mutated clones remains time-consuming, laborious and/or expensive.<h4>Results</h4>Here we report a high-throughput screening strategy that allows parallel screening of up ...[more]

PMID: 25409780

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Project description:Currently available methods for the laboratory investigation of Legionella pneumophila outbreaks require organism culture. The ability to sequence L. pneumophila directly from clinical samples would significantly reduce delays. Here, we develop a method for targeted next-generation sequencing (NGS) of selected L. pneumophila genes utilizing a CRISPR/Cas9-based target enrichment system. We determine the method's utility by typing cultured L. pneumophila isolates and subsequently apply the method directly to patient samples. We sequenced 10 L. pneumophila isolates by 2 methods, (i) whole-genome sequencing (WGS) and (ii) targeted (CRISPR/Cas9-based) finding low-abundance sequences by hybridization (FLASH)-NGS, sequencing 57 selected genes. The targeted NGS of 57 genes was more efficient than WGS, and phylogenetic analysis of the 57 genes yielded the same classification of the L. pneumophila isolates as that based on analysis of whole-genome data. Furthermore, targeted NGS of L. pneumophila performed directly on patient respiratory samples correctly classified the patients according to their corresponding cultured isolates. This provides proof of concept that targeted NGS can be used to sequence L. pneumophila directly from patient samples. Studies on a larger number of patient samples will further validate this method. Nonetheless, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms. IMPORTANCE The bacterium Legionella pneumophila is responsible for outbreaks of serious and life-threatening pneumonia called Legionnaires' disease. There is a need for new molecular methods that allow investigation of Legionella outbreaks directly from patient samples, without the need for prior microbiological culture, which causes delays. Our study aims to address this problem. We have utilized a CRISPR/Cas9-based targeted next-generation sequencing (NGS) method that can be applied directly on human specimens. Furthermore, we show that analysis of the sequences of a small number of targeted genes offers the same classification of L. pneumophila as that based on data derived from the whole genome. Given the rising interest globally in sequencing pathogens directly from human samples, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms.

Dataset Information

A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing.

Publications

A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing.

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