Preservation system using Cas9
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). Synthetically chimeric single stranded guide RNA (sgRNA) was designed by combining crRNA and tracrRNA. In the sgRNA, 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). The resulting double strand break introduced by Cas9 leads to exonucleolytic degradation of the DNA in prokaryotic cells (Simmon and Lederberg, 1972).
In our case we envision a retention system, where Cas9 cleaves Plasmids at sites where the UBP is absent. This works by using a sgRNA complementary to the DNA sequence without the UBP. In plasmids with the UBP present, the mismatch between isoG/isoCm and sgRNA greatly decreases Cas9 activity (Zhang et al., 2017). In the event of UBP loss, the sgRNA now binds perfectly to the mutated site and restores Cas9 activity which leads to degradation of the mutated plasmid. Consequently, this leads to a retention of the UBP in the plasmids.
Figure 1: UBP conservation system using Cas9.
sgRNAs are targeted against every DNA sequence emerging from UBP loss on a plasmid. A: Loss of the UBP leads to a point mutation. Now a sgRNA 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 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 + PlacO‑tight1 (BBa_K2201024) and terminator + RFS (BBa_K2201025) that can be assembled with any coding sequence 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 PlacO‑tight1.
Optimization of the negatively regulating promoter Plac
Figure 3: DNA map of the wild type lac operon and its transcriptional and translational products (Oehler et al., 1994).
Figure 4: The wild type lac operators and its regulatory structures.
The tetrameric Lac repressor can bind either the lac operators O1 and O3 or O1 and O2 to form a DNA loop. The DNA loops efficiently inhibit the transcription by the CAP protein (Oehler et al., 1994).
Figure 5: The lac operator Oid.
The inversion is indicated by the arrow for the perfectly symmetric lac operator Oid. It is the inverted repeat of the left half of O1 (Sadler et al. 1983).
Figure 6: Repression values dependent on inter‑operator distances between O1 and Oid.
The repression values refer to the repression of the chromosomal lacZ gene under the control of O1 at its natural position and Oid at the indicated position. With 50 tetrameric Lac repressors per cell the repression value is calculated by the specific activity of β‑galactosidase in absence of active Lac repressor divided by the specific activity of β‑galactosidase in the presence of active Lac repressor. The dashed line shows the repression value for a single natural O1 operator (Mueller et al., 1996).
According to these results, our PlacO‑tight1 consists of the auxiliary operator Oid with a 70.5 bp spacing to O1 at its natural position. The residuray sequence like the Plac was kept as in the natural lac operon taken from E. coli BL21(DE3) (Figure 7).
Figure 7: Tight lac operon PlacO‑tight1.
The figure with its annotations was created with the software Geneious 10.0.8.
Deletion of codA
Figure 8: Reactions catalyzed by the cytosine deaminase.
A) The conversion of cytosine to uracil is a normal reaction step within the pyrimidine metabolism. B) Isocytosine is converted into uracil by the cytosine deaminase. C) Isoguanine is formed during oxidative stress. Cytosine deaminase catalyzes the reaction to the non-mutagenic xanthine.
Figure 9: Arrangement of the codBA operon.
codA is located downstream of codB and 1.3 kb in size. Both genes overlap by 11 bases. codB codes for cytosine permease, while codA codes for cytosine deaminase.
Figure 10: Crystal structure of cytosine deaminase from Escherichia coli complexed with zinc and phosphono-cytosine.
The structure was determined by X-Ray crystallography with a resolution of 1.71 Å (Hall et al., 2011).