Fig. 5: Repurposing the endogenous type I-C CRISPR-Cas system in E. lenta enables markerless deletion of dadR. | Nature Communications

Fig. 5: Repurposing the endogenous type I-C CRISPR-Cas system in E. lenta enables markerless deletion of dadR.

From: Genetic manipulation of the human gut bacterium Eggerthella lenta reveals a widespread family of transcriptional regulators

Fig. 5

a Architecture of the CRISPR-Cas locus in E. lenta DSM 2243. b Spacer choice for dadR-targeting crRNA and optimization of cumate-inducible crRNA construct. c Induction of dadR-targeting crRNA (gDadR) decreased the CFUs when compared to an uninduced control. No significant difference in CFUs was observed when the NT was induced. A single representative image is shown with n = 3 biological replicates. d Repair template is introduced to make editing plasmid pXD71Cas10.1RT. To probe potential dadR deletion, primers flanking the repair template were used. e Workflow for generating the ΔdadR deletion strain. f PCR screening of colonies formed after initial pXD71Cas10.1RT transformation revealed most colonies contained both non-edited (4 kb) and edited cells (2.5 kb). g PCR screening of colonies formed after spreading initial pXD71Cas10.1RT transformant cultures onto a cumate-containing plate revealed clean ΔdadR mutants (2.5 kb), which was further confirmed by Sanger sequencing. Experiments shown in panels f and g were performed once on randomly selected colonies. Source data are provided as a Source Data file.

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