Fig. 6: Timetable of full NT-CRISPR procedure and plasmid curing.

a Under ideal conditions, eight to ten hours per day are sufficient to go from preparation of tDNAs and cloning of gRNAs into the NT-CRISPR plasmid to a plasmid-cured edited strain in one week (Monday to Friday). Steps marked with asterisks (verifiction of edits by cPCR and Sanger sequencing, as well as the confirmation of plasmid loss after plasmid curing) can be performed in parallel with the next steps. To increase success rate, multiple colonies can be used for the consecutive step and later be discarded if verification yields negative results. Details for all individual steps can be found in the Method sections. b Plasmid curing efficiency. The workflow was performed as described in the Method sections with colonies resulting from deletion of wbfF with NT-CRISPR. n = 20 replicates, representing five wbfF deleted colonies of two transformants of the NT-CRISPR plasmid (circle or triangle) and two independent experiments (filled or open symbols). The bar shows the mean of all replicates and the error bar indicates the standard deviation of the mean. The reported fraction of plasmid-cured colonies is the result of streaking 20 colonies of each replicate on LBv2 agar plates with and without chloramphenicol. Colonies resulting in growth on LBv2 agar plates without but not with chloramphenicol were considered to consist of cells that have lost the NT-CRISPR plasmid. Fotos of streaked colonies are shown in Supplementary Fig. S11.