Re-sensitizing bacteria to antibiotics and plasmid editing by Sh95 I-F1 CRISPR-Cas

MARÍA CAROLINA MOLINA; CECILIA QUIROGA

New York

Congreso; Genome Engineering: CRISPR Frontiers; 2023

Cold Spring Harbor Laboratory

In nature, CRISPR-Cas systems exist with an extraordinary diversity ofgenetic structures and mechanisms of immunity against invasion by mobilegenetic elements. In the last years, multiple efforts focused to transfer thesenatural systems to different organisms and redirect their activity towardsspecific genes leading to major advances in the gene editing field. In thiswork, we studied the I-F1 CRISPR-Cas system from the clinical isolateShewanella xiamenensis Sh95 for endogenous reprogramming usingspecifically designed guides provided in trans and sought to transfer itsinterference machinery (Sh95 I-F1CRISPR-Cas) to Escherichia coli.Endogenously, S. xiamenensis Sh95 was transformed with plasmidsexpressing anti-gfp or control guides and with a plasmid harboring gfp(pBKgfp KmR) in a subsequent step. Interference over pBKgfp showed a~3-fold log CFU/mL reduction for anti-gfp vs. control guide. In a secondapproach, by introducing anti-gfp targeting guides into S. xiamenensisSh95/pBKgfp we were able to recover plasmid-cured KmS colonies. Theefficiency of transformation showed ratios of selective versus non-selectiveplates (NS/S) of 22.4 log CFU/mL for the interference assays and 21.4 logCFU/mL for the curing assays, values over 10 times higher than controls(NS/S = 2 and 2.2 log CFU/mL). Loss of fluorescence intensity wasevaluated by fluorescence microscopy and fluorimetry. Plasmid sequenceelimination was confirmed by PCR screening. Evaluation of Sh95 I-F1CRISPR-Cas activity transferred to E. coli BL21(DE3) resulted in NS/Sratios of 2.3 log CFU/mL when targeting a gene under no selective pressure(gfp) and 17.2 log CFU/mL when targeting blaTEM, conferring resistance toampicillin. As for control guides, NS/S ratios were 1.4 log CFU/mL. Next,three independent experiments were performed, survival colonies wererandomly picked and further analyzed by fluorimetry, fluorescencemicroscopy, and flow cytometry. Measures and observation of fluorescenceintensity showed a reduction to reference WT levels. At the sequence level,different plasmid editing outcomes were detected by PCR screening andconfirmed by Sanger sequencing, with sizes ranging from Δ243bp, Δ~3kbp,Δ~4kbp, showing a bidirectional degradation pattern. The analysis revealedthat these colonies suffered different plasmid editing events that yieldedsmaller molecules. Overall, we were able to confirm the activity of thissystem and re-sensitize S. xiamenensis Sh95 and E. coli to kanamycin(Δaph3´) and ampicillin (ΔblaTEM), respectively. Finally, our resultsdemonstrate the practical use of Sh95 I-F1CRISPR-Cas as a gene editing tool.