Fig. 2: Schematic overview of the RECKLEEN system’s workflow.
The RECKLEEN workflow started by inserting annealed oligonucleotides into the RECKLEEN plasmid, generating a 20-nt spacer sequence in the sgRNA that precisely targets the protospacer sequence in the target gene of Kp genome. This plasmid was then introduced into Kp by electroporation. Next, the recovered cells were cultured in presence of IPTG, to induce the Ptac promoter for the expression of the lambda Red operon genes. Then, these cells were employed to prepare electrocompetent cells. Subsequently, donor DNA (dDNA) template containing the homology arms for the target gene is then electroporated into these electrocompetent cells. The cells were then recovered in S.O.C. medium for 3 h. The recovered cells were then incubated overnight on plates containing the inducer ATc. During this period, some of the cells had the opportunity to undergo the recombination event, facilitated by the lambda Red components. Concurrently, ATc induces the Ptet promoter to overexpress the cas9/sgRNA components enabling the counterselection step by specifically targeting and cleaving the chromosome of the WT cells. Only cells that successfully underwent the recombination event were able to evade the lethal Cas9/sgRNA-induced DSB, allowing them to grow on the plates, as they no longer contained the target sequence. After confirming the accuracy of the edits, the RECKLEEN editing plasmid was cured, allowing the plasmid origin and resistance marker to be reused in subsequent experiments if needed. Detailed description of the RECKLEEN system’s workflow is provided in “Methods” section.
