Introduction

Ave Caesar

The Idea

To prove our hypothesis, we planned a circuit for the directed evolution of PAM specificity of Cas9 (Fig. 1). The main challenge was to link transcription activation to the binding of Cas9. We chose a system, which contains a dCas9 fused to a RNA polymerase Ω subunit (rpoZ) BIKARDETAL..2013. The rpoZ is able to recruit the transcription machinery to a poromoter and therefore activate gene expression. In our scenario, the nuclease targets a region upstream of a minimal promoter. In case the dCas9 is able to bind the DNA, the fused rpoZ activates gene expressio expreession. The J23117 promoter from the registry was chosen for activation BIKARDETAL..2013. It stands our through its low background activity and can be activated by the factor of 23 when rpoZ binds. Upstream of the promoter sequence, a spacer with a wildtype NGG PAM, that was previously used to target RFP was placed QIETAL..2013. If the Cas9 is able to bind to the spacer, transcription is activated. By changing the PAM sequence or generating PAM libraries, it is possible to induce a selection pressure on the randomly mutating protein. As a result, proteins with a weaker PAM specificity evolve, which can be used, no matter if the NGG motif is present exactly at the desired position. Of course, this circuit was designed according to our cloning standard by Gibson assembly.

Phage Based in vivo Eolution with GeneVI

One of the major challenges in the context of transcription activation with help of the rpoZ is leaky expression. This is a setious problem for PACE, because if geneIII is expressed prior to phage infection leads to infection the bactirial cell turns resistant RN44. Fortunately, it was shown that geneVI can be used for phage propagation in the context of directed evolution as well. In contrast to the commonly used geneIII, it has the advantage that leaky geneVI expression has no effect on the infectivity of E. coli by phages BRODELETAL..2016. As a consequence, we decided to adapt this approach and towork with geneVI instead of geneIII. All parts, which were necessary for the assembly of Accessory Plasmids were generated by PCR with the respective homology regions in the extensions. Subsequently, they were assembled by Gibson assembly (Fig.:2). All APs carry a bicistronic operon for the expression of geneIII and luxAB as luminescent reporter downstream of the promoter, described above. An expression cassette with the required gRNA under the control of a constitutive promoter is located on the same plasmid. APs varying in the copy number of their origins of replication and the strength of the RBS upstream of geneVI were cloned. To evolve the PAM specificity, we generated PAM libraries with four randomized nucleotides next to the spacer sequence. In order to do so, the whole plasmid was PCR amplified with the four PAM nucleotides as primer extensions. Subsequently, the plasmid was reassembled by Golden Gate assembly. To avoid that nucleotides, that pair with the original PAM are preferred and overrepresented in the library, a BbsI site was inserted next to the PAM. Prior to the PCR, the plasmid was digested with the enzyme, resulting in a linear fragment. The four nucleotides were loceated in overhangs, in the strand, to which the primer cannot bind. Plasmids that were cloned for the evolution of PAM specificity, the plasmid names, and the functional parts they consist of are shown in (Tab.: 1).