Part:BBa_K2201111

crRNA R-target mutA

crRNA R-target mutA

Usage and Biology

crRNAs are the RNA sequences of sgRNAs that are responsible for binding the target sequence. Normally, they are 20 nt long, targeting a 20 nt target sequence upstream of a proto-spacer adjacent motif (PAM) Sequence (5’-NGG-3’). Cas9 nuclease will cleave approximately 3 bases upstream of the PAM.

This part was designed and constructed to be used in a preservation system for unnatural base pairs using Cas9. The composite part used for the respective experiments is BBa_K2201032

For this specific use case, we designed the crRNAs to be 18 nt long, reducing its binding strength in order to increase the influence of the mismatch produced by the unnatural basepair in the target sequence. Only if the unnatural base pair is absent due to mutation to a natural base, the crRNA will be able to bind its target.

Preservation system using Cas9

Due to tautomerisation of isoG and hydrolysis of isoC^m and the resulting loss of the unnatural base pair (UBP), there is a need for a system, to preserve the UBP on the plasmid. In 2017, Zhang et al. successfully deployed a CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 system for retention of a UBP. We adapted this conservation system to our UBP and thus used CRISPR/Cas9 to eliminate all plasmid DNA that had lost the UBP.
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/isoC^m 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.

References

Ran, F.A., Hsu, P.D., Lin, C., Gootenberg, J.S., Konermann, S., Trevino, A.E., Scott, D. a, Inoue, A., Matoba, S., Zhang, Y., and Zhang, F. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154: 1380–9.

Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., and Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8: 2281–2308.

Simmon, V.F. and Lederberg, S. (1972). Degradation of bacteriophage lambda deoxyribonucleic acid after restriction by Escherichia coli K-12. J. Bacteriol. 112: 161–9.

Zhang, Y., Lamb, B.M., Feldman, A.W., Zhou, A.X., Lavergne, T., Li, L., and Romesberg, F.E. (2017). A semisynthetic organism engineered for the stable expansion of the genetic alphabet. Proc. Natl. Acad. Sci. 114: 1317–1322.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]