The nuclease Cas9 is part of the adaptive immune system of Streptococcus pyogenes, where it induces double strand breaks in the genomic DNA. This enzyme is recruited by a CRISPR RNA (crRNA). A crRNA consists of direct repeats interspaced by variable sequences called protospacer. Those protospacers are derived from foreign DNA and encode the Cas9 guiding sequence (guide RNA). An auxiliary transactivating crRNA (tracrRNA) helps processing the precursor crRNA array into an active crRNA that contains the 20 nucleotide guide RNA. The guide RNA binds to the complementary genomic DNA sequence via Watson‑Crick base pairing. For this binding, the genomic DNA sequence needs to be located upstream of a CRISPR type II specific 5’ NGG protospacer adjacent motif (PAM). To combine crRNA and tracrRNA a chimeric single stranded guide RNA (sgRNA) was designed. Therefore, only the 20 nucleotide guiding sequence needs to be exchanged for targeting any genomic sequence followed by a PAM sequence (Ran et al., 2013 a, b). A double strand break introduced by Cas9 leads to DNA degradation by exonucleases in prokaryotic cells (Simmon and Lederberg, 1972).
The UBP isoG and isoCm is an orthogonal system. UBP inside the target DNA causes a mismatch to the sgRNA generally reducing the cleavage activity of Cas9 (Zhang et al., 2017). Accordingly, Cas9 can be programmed by sgRNAs to cleave all plasmids, which had lost the UBP due to point mutations (Figure 1). Consequently, this leads to a retention of the UBP in the plasmids.
Figure 1: UBP conservation system using Cas9.
sgRNAs are targeted against every possible DNA sequence that had lost the UBP, which was incorporated on a plasmid. A: The UBP gets lost, which leads to a point mutation. One of the sgRNAs can bind to the DNA target sequence. Cas9 is recruited and cleaves the plasmid, which is followed by its degradation. B: Plasmids that contain a UBP in the DNA target sequence lead to a mismatch with every sgRNA. Cas9 does not cleave the plasmid, leading to a retention of the UBP.
Figure 2: The repair template (BBa_K2201028) for genomic integration of cas9 into E. coli BL21(DE3).
The 1 kb left flanking sequence (LFS: BBa_K2201021) and right flanking sequence (RFS: K2201022) are taken from the genome of E. coli BL21(DE3). Inside the genome LFS and RFS are directly flanking the coding sequence of arsB. The strong terminators from BBa_B0015 were used to avoid basal expression and to stop transcription right after cas9. There are composite parts the consist of LFS + terminator + Plac‑tight (BBa_K2201024) and terminator + RFS (BBa_K2201025) that can be assembled with any coding seqeunce of interest to create a repair template. The coding sequence of cas9 is taken from the pCRISPomyces plasmid system by Cobb et al., 2014 and is originated from S. pyogenes. It is negatively regulated by the IPTG‑inducible promoter Plac‑tight.