Part:BBa_K4212007

Cas9

Codon optimised Cas9 CDS for B. subtilis. Useful for CRISPR-Cas9 experiments in B. subtilis. Used in the Sporadicate project to develop a self-digesting plasmid system.

Usage and Biology

Cas9 is a large protein, consisting of 1368 residues ( ≈4.1kb). Its synthesis from IDT as a single linear fragment was ruled out due to the complexity of the build. For this reason, we once again relied on customized overhangs which split the protein CDS’s in half to allow for a directional and scarless assembly. In particular, these would come together and reconstitute the CDS in a L0 vector (STK001). Then, by harnessing the destination backbone 1C and 0A spacer, Cas9 would be paired with the appropriate regulatory elements (RBS and terminator) to allow for its translation. Indeed, Cas9’s construction in 1C lacks a promoter as the transcriptional levels are regulated upstream by PsspB (1B vector).(image)Notably, cas9 was the largest protein we attempted to clone across Sporadicate. As soon as linear fragments were obtained and resuspended at 50fmol/μL in water, we tested two different molar ratios of vector to insert (1:5 and 1:3) in L0 Golden Gate assemblies. This choice is motivated by the attempt to drive assembly of Cas9 in the destination backbone (1C) by enhancing the pool of CDS inserts in the reaction mix.

Figure 1. The schematic design of Cas9

References

[1] Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A. & Zhang, F. (2013) Genome engineering using the CRISPR-Cas9 system. Nature Protocols. 8 (11), 2281–2308. doi:10.1038/nprot.2013.143. [2] Zhang, F., Wen, Y. & Guo, X. (2014) CRISPR/Cas9 for genome editing: progress, implications and challenges. Human Molecular Genetics. 23 (R1), R40–R46. doi:10.1093/hmg/ddu125.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 3823
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3823
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3823
  Illegal BglII site found at 1188
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 3823
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 3823
  
• 1000
  COMPATIBLE WITH RFC[1000]