Reversal of carbapenem-resistance in Shewanella algae by CRISPR/Cas9 genome editing
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Reversal of carbapenem-resistance in Shewanella algae by CRISPR/Cas9 genome editing based on the original paper by Wu, Z., Huang, Y., Chao, W., Ho, S., Cheng, J. and Liu, P. (2019). Journal of Advanced Research, 18, pp.61-69
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Reversal of carbapenem-resistance in Shewanella algae by CRISPR/Cas9 genome editing
- 1. Reversal of carbapenem- resistance in Shewanella algae by CRISPR/Cas9 genome editing Wu, Z., Huang, Y., Chao, W., Ho, S., Cheng, J. and Liu, P. (2019). Journal of Advanced Research, 18, pp.61-69
- 2. Reversal of carbapenem-resistance in Shewanella algae What’s the relevance of this study? • Carbapenem antibiotic resistance is serious • Shewanella algae is ubiquitous in marine environments globally • Reservoir of resistance in the environment and in humans • 20,000 deaths in US and 25,000 in EU annually • Many $billions in healthcare expenditureCarbapenem family
- 3. What is Shewanella? • Gram-negative, motile, facultative anaerobic marine bacillus • Causes bacteraemia, soft tissue and intra-abdominal infections • Open wounds particularly susceptible • Contracted through immersion in seawater or raw seafood
- 5. Re-sensitisation - Method • Phase I: Genome sequencing, assembly, annotation • Phase II: Detection of antibiotic resistance genes and target selection • Phase III: Targeted gene deletion using CRISPR/Cas9 • Phase IV: Screening of targeted gene deletion + antimicrobial susceptibility testing
- 6. Phase I: Genome sequencing, assembly, annotation DNA sequence contig assembly Culture S. algae VGH117 Extract DNA Sequence DNA Annotation + BLAST analysis
- 7. Phase II: Detection of antibiotic resistance genes and target selection Antibiotic-resistance genes predicted using Comprehensive Antibiotic Resistance Database (CARD, https://card.mcmaster.ca/home) bla-OXA-55-like gene NmcR-like gene Sul2 gene
- 8. Phase III: CRISPR/Cas9 • Construction of the Cas9 containing plasmid • Design and addition of targeted sgRNA • Co-culture of CRISPR/Cas9 plasmid and Shewanella • Targeted gene deletion “product”
- 9. Phase IV: Screening of targeted gene deletion + antimicrobial susceptibility testing PCR E-test
- 10. Results Sul2 gene KO Bla-OXA-55- like gene KO Bla-OXA-55- like gene KO NmcR-like gene KO – no difference to wild-type
- 11. Discussion • Successful re-sensitisation by CRISPR/Cas9 genome editing. • Identification of the essential gene for carbapenem resistance in S. algae. • CRISPR/Cas9 genome editing is a promising approach to validate gene function But • Is this study a drop in the resistance ocean? • How do we deliver therapeutically, safely, ethically? • What about the environmental reservoir?
- 12. Thank you.
Editor's Notes
- Carbapenem – critical importance list of WHO List of Essential Medicines Global spread of carbapenem resistance Shewanella “emerging” pathogen
- Nanowires
- 1. Culture vgh117 = human clinical isolate grown on agar @35C for 12 hours 2. DNA extracted - quality and quantity using Nanodrop and Qubit fluorometer 3. Chopped up/sheared and Into Pacific Biosciences RSII sequencer 4. The postfiltered reads were assembled by Canu (v1.4) [21], which produced one single large chromosomal contig + plasmid 5. Protein-coding and non-coding genes in the VGH117 genome and plasmid were annotated using the National Center for Biotechnology Information (NCBI) Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) And BLAST analysis to get functional characteristics of the annotated genes
- The comprehensive resistance database. Open source, originated from Canada but curated and contributed by worldwide collaborators Cross reference homologous genes from same species to even different genuses 3 candidate genes selected
- CRISPR/Cas 9 derived from system of adaptive immunity in bacteria protecting its own DNA from invading viruses/plasmids via RNA-guided DNA cleavage by Cas proteins Short segments of foreign DNA are integrated within the CRISPR locus and transcribed into CRISPR RNA (crRNA), which then anneal to trans-activating crRNA (tracrRNA) to direct sequence-specific degradation of pathogenic DNA by the Cas9 protein now consists of only the Cas9 nuclease and a single guide RNA (gRNA) Use same principle to degrade target DNA sequence CAS9 nuclease from strep pyogenes, engineered gRNA plus a few other building blocks cloned into pCC1fosmid vector backbone. Cobbled together within E.coli and transferred to another E.coli conjugation strain E.Coli plus shewanella then mixed and cultured.
- ZFN – zinc finger nucleases lack target specificity as cannot recognize all nucleotide triplets But – clin trials still ongoing in HIV and haemophilia B. TALEN – transcription activator like effector nucleases – repetitive structure means bigger molecule = diff to introduce vua viruses