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Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes.

ABSTRACT: Unraveling the mechanism of action and molecular target of small molecules remains a major challenge in drug discovery. While many cancer drugs target genetic vulnerabilities, loss-of-function screens fail to identify essential genes in drug mechanism of action. Here, we report CRISPRres, a CRISPR-Cas-based genetic screening approach to rapidly derive and identify drug resistance mutations in essential genes. It exploits the local genetic variation created by CRISPR-Cas-induced non-homologous end-joining (NHEJ) repair to generate a wide variety of functional in-frame mutations. Using large sgRNA tiling libraries and known drug-target pairs, we validate it as a target identification approach. We apply CRISPRres to the anticancer agent KPT-9274 and identify nicotinamide phosphoribosyltransferase (NAMPT) as its main target. These results present a powerful and simple genetic approach to create many protein variants that, in combination with positive selection, can be applied to reveal the cellular target of small-molecule inhibitors.

SUBMITTER: Neggers JE

PROVIDER: S-EPMC5799254 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes.

Neggers Jasper Edgar JE Kwanten Bert B Dierckx Tim T Noguchi Hiroki H Voet Arnout A Bral Lotte L Minner Kristien K Massant Bob B Kint Nicolas N Delforge Michel M Vercruysse Thomas T Baloglu Erkan E Senapedis William W Jacquemyn Maarten M Daelemans Dirk D

Nature communications 20180205 1

Unraveling the mechanism of action and molecular target of small molecules remains a major challenge in drug discovery. While many cancer drugs target genetic vulnerabilities, loss-of-function screens fail to identify essential genes in drug mechanism of action. Here, we report CRISPRres, a CRISPR-Cas-based genetic screening approach to rapidly derive and identify drug resistance mutations in essential genes. It exploits the local genetic variation created by CRISPR-Cas-induced non-homologous en ...[more]

PMID: 29402884

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Project description:BackgroundThe exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a "brute force" validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions.ResultsWe first set up our own version of the CRISPR/Cas9 protocol and then we evaluated the mutagenesis efficiency by changing different parameters including sequence of guide RNAs, length and concentration of donor DNAs, and use of single stranded and double stranded donor DNAs. We then validated the optimized conditions targeting 78 "dispensable" genes. This work led to the definition of a protocol, featuring the use of double stranded synthetic donor DNAs, which guarantees mutagenesis efficiencies consistently higher than 10% and a robustness of 100%. The procedure can be applied also for simultaneous gene deletions.ConclusionsThis work defines for the first time the robustness of a CRISPR/Cas9-based protocol based on a large sample size. Since the technical solutions here proposed can be applied to other similar procedures, the data could be of general interest for the scientific community working on bacterial genome editing and, in particular, for those involved in synthetic biology projects requiring high throughput procedures.

Dataset Information

Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes.

Publications

Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes.

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