Enhanced killing of antibiotic-resistant bacteria enabled by massively parallel combinatorial genetics

Cheng, Allen A.; Ding, Huiming; Lu, Timothy K.

doi:10.1073/pnas.1400093111

Cited by 36 publications

(27 citation statements)

References 57 publications

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“…The proposed mechanism relies on an increase in dsDNA breaks leading to activation of the SOS response and antibiotic independent cell death [Citorik 2014]. A combinatorial TR overexpression screen in bla NDM-1 E. coli identified many gene pairs that induced lethality in the presence or absence of ceftriaxone [Cheng 2014]. These results support the notion that antibiotic cytotoxicity is a result of target specific inhibition coupled to increased redox activity via a global stress response [Dwyer 2014].…”

Section: Treatment Optionsmentioning

confidence: 82%

The rapid spread of carbapenem-resistant Enterobacteriaceae

Potter

D’Souza

Dantas

2016

Drug Resistance Updates

298

205

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Carbapenems, our one-time silver bullet for multidrug resistant bacterial infections, are now threatened by widespread dissemination of carbapenem-resistant Enterobacteriaceae (CRE). Successful expansion of Enterobacteriaceae clonal groups and frequent horizontal gene transfer of carbapenemase expressing plasmids are causing increasing carbapenem resistance. Recent advances in genetic and phenotypic detection facilitate global surveillance of CRE diversity and prevalence. In particular, whole genome sequencing enabled efficient tracking, annotation, and study of genetic elements colocalized with carbapenemase genes on chromosomes and on plasmids. Improved characterization helps detail the co-occurrence of other antibiotic resistance genes in CRE isolates and helps identify pan-drug resistance mechanisms. The novel β-lactamase inhibitor, avibactam, combined with ceftazidime or aztreonam, is a promising CRE treatment compared to current colistin or tigecycline regimens. To halt increasing CRE-associated morbidity and mortality, we must continue quality, cooperative monitoring and urgently investigate novel treatments.

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Section: Treatment Optionsmentioning

confidence: 82%

The rapid spread of carbapenem-resistant Enterobacteriaceae

Potter

D’Souza

Dantas

2016

Drug Resistance Updates

298

205

View full text Add to dashboard Cite

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“…Despite its simplicity for multiplexed genetic perturbations (10)(11)(12), new methods are needed to enable highthroughput CRISPR-Cas9-based screening with combinatorial sets of guide RNAs (gRNAs), which would be broadly useful for studying combinatorial gene functions in multigenic phenotypes and diseases. By using CombiGEM-based DNA assembly (13,14), we developed a strategy for the simple and efficient assembly of barcoded combinatorial gRNA libraries. These libraries can be delivered into human cells by lentiviruses to create genetically ultradiverse cell populations harboring unique gRNA combinations that can be tracked via barcode sequencing in pooled assays.…”

mentioning

confidence: 99%

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

Wong

Choi

Cui

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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214

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The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (Combi-GEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas-based knockouts and RNA-interference-based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.CRISPR-Cas | CombiGEM | multifactorial genetics | genetic perturbations | high-throughput screening

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“…2b). Greater coverage in the libraries could be attained by scaling up library transformations and increasing the number of sequencing reads per sample 30 . Such efforts could help to increase coverage of the missing three-wise combinations (~11% of the total expected combinations) in the plasmid library.…”

Section: Resultsmentioning

confidence: 99%

Massively parallel high-order combinatorial genetics in human cells

et al. 2015

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The systematic functional analysis of combinatorial genetics has been limited by the throughput that can be achieved and the order of complexity that can be studied. To enable massively parallel characterization of genetic combinations in human cells, we developed a technology for rapid, scalable assembly of high-order barcoded combinatorial genetic libraries that can be quantified with high-throughput sequencing. We applied this technology, combinatorial genetics en masse (CombiGEM), to create high-coverage libraries of 1,521 two-wise and 51,770 three-wise barcoded combinations of 39 human microRNA (miRNA) precursors. We identified miRNA combinations that synergistically sensitize drug-resistant cancer cells to chemotherapy and/or inhibit cancer cell proliferation, providing insights into complex miRNA networks. More broadly, our method will enable high-throughput profiling of multifactorial genetic combinations that regulate phenotypes of relevance to biomedicine, biotechnology and basic science.Complex biological traits are regulated by the concerted action of combinatorial gene sets 1 . For example, multiple genetic factors are needed to reprogram somatic cells into induced pluripotent stem cells or distinct cell types 2 . Combinatorial drug therapies can achieve enhanced efficacy over conventional monotherapies, because targeting multiple pathways can be synergistic 3 . Furthermore, although genomewide association studies have implicated multiple individual loci in multifactorial human diseases, these loci can explain only a minor Reprints and permissions information is available online at

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Enhanced killing of antibiotic-resistant bacteria enabled by massively parallel combinatorial genetics

Cited by 36 publications

References 57 publications

The rapid spread of carbapenem-resistant Enterobacteriaceae

The rapid spread of carbapenem-resistant Enterobacteriaceae

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

Massively parallel high-order combinatorial genetics in human cells

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