Part:BBa_K3904401

CRISPR-Cas9 is a versatile genome enditing technique. In our approach to edit E. coli Nissle 1917 genome, we have used two plasmid based system enableing to combine Lambda Red recombination and CRISPR-Cas9 as cunterselection tool - pCas (could be found in addgene: #62225) and pTarget (could be found in addgene as pTargetF: #62226).

Mechanism of genome editing pCas plasmid is used for Cas9, Lambda Red system expression and plasmid curing of pTarget. Cas9 - the RNA-guided endonuclease - is expressed constitutively, while the expression of Lambda Red genes (Gam, Exo, Beta) is under the control of arabinose inducable promoter araBp. pTarget plasmid caries consitutively expressed signle-guide RNA (sgRNA). This RNA molecule, as and in nature, is composed of two central parts: crispr RNA (crRNA) and tracrRNA. crRNA is 17-20 nt lenght RNA sequence complementary to the targeted DNA adjecent to the protospacer adjacent motif (PAM) and tracrRNA is the scaffold for the Cas (in this case Cas9) nuclease binding to guide RNA and forming the ribonucleoprotein complex (1). In nature those two parts exists as two seperate RNA molecules, however, in laboratory experiments they are usually combined into one single-guide RNA (sgRNA). As both pCas and pTarget plasmids are in cell, Cas9 nuclease and sgRNA are able to form ribonucleoprotein complex and perform double strand break in the chosen part of the DNA. However, if arabinose has been added to the cell culture and double stranded DNA repair tamplet is present in the cell, Lambda Red system performs homologous recombination. If this process is unsuccessful, Cas9-sgRNA complex will cause double strand break and will cause cell death (2). This is employed as a counterselection in order to avoid of the additional antibiotic as selection marker usage.

Plasmid curing pCas also caries sgRNA targeting pTarget plasmid. This sgRNA is expressed under the controle of IPTG inducable lac operon. As IPTG added to the culture media it binds repressor lacI and allows to begin transcription of sgRNA targeted to pTarget replicon. pCas plasmid is cured by growing overnight culture at 37°C.