Part:BBa_K1903002:Design

dCas9 with protein insertion frames version B

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1046
  Illegal BglII site found at 2822
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 3536
  Illegal SapI.rc site found at 1159
  

Design Notes

This dCas9 includes a Double Terminator and has two frames that enable the insertion of proteins in the amino acids N588 and N888. With structural modeling we found that this two hotspots come close when the dCas9 binds to the sgRNA, which could trigger a reaction between the proteins inserted in the sites.

dCas9 version B has the insertion frames from amino acids 583 to 601 and from 882 to 908 and each frame has BbsI recognition sites at its start and end; the "start" recognition sites from each insertion frame are inverted so that the four recognition sequences can be lost after the Golden Gate reaction, leaving a functional Cas9 with two protein insertions. As its shown below, every frame has different fusion sites: the first frame is limited by GGAG and GCTT sites and the second one by AATG and TACT; all these sites are part of the Cas9 protein sequence and are going to be kept after the insertions's assembly.

We designed two protein insertions for this dCas9 which can be found as BioBricks BBa_K1903022 and BBa_K1903023. Using this BioBricks sequences as a temple, you can design two different protein insertions for the dCas9 version A.

We successfully assembled our dCas9 (without insertions) and verified in this agarose gel. The dCas9 is a approximately of 4500 bp

Note: Because our dCasB has four BbsI recognition sites in its sequence, it is not a functional protein until the two insertions are assembled and the recognition sites's sequences are lost.

References

Oakes, B. L., Nadler, D. C., Flamholz, A., Fellmann, C., Staahl, B. T., Doudna, J. A., & Savage, D. F. (2016). Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch. Nature biotechnology, 34(6), 646-651

Sternberg, S. H., LaFrance, B., Kaplan, M., & Doudna, J. A. (2015). Conformational control of DNA target cleavage by CRISPR–Cas9. Nature, 527(7576), 110-113.

Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PloS one, 6(2), e16765.