Part:BBa_K2130000

∆RuvCIII-2 ∆HNH Sp-dCas9

dCas9 is a catalytically inactive Cas9, which still retains it's ability to bind to DNA. For epigenetic regulation, the dCas9 is highly dependent upon the function of it's fusion partner.

Usage and Biology

For this part, we have deleted the HNH domain as well as the RuvCIII-2 region of the SP-dCas9 and tested it's binding capability by assessing the strength in transcriptional activation via by tagging it with a VP64-p65-Rta tripartite activator. A GFP reporter, BBa_K2130003 with two dCas9 binding site was used to measure the strength of the truncated dCss9.

Conformation of dCas9 before and after non-targeted strand cleavage. In state B, there are several regions (highlighted purple in state A), that are unresolved which points out that these regions are very dynamic and is not important for DNA interaction. We named this dynamic region as RuvCIII-2. On the other hand, RuvCIII-1(colored in cyan) also has minimal contact with DNA.

Our results showed that RuvCIII-2 which is highly dynamic can be deleted while RuvCIII-1 couldn't. RuvCIII-2 deletion can be combined with the HNH deletion producing a truncated version of more than 60% of activity.

We then compared our ∆HNH∆RuvCIII-2 dCas9 with other truncation. This part still has stronger activation strength than the ∆REC2∆HNH mutant although with an extra of ~200bp.

Improvement

Team NTU_SINGAPORE 2017 made an improvement to this part by exploring possibilities to further shrink the size of dCas9 into part BBa_K2316000. Furthermore, they even demonstrated that the improved version can be used in CRISPRi system.

References

VPR tripartite activator:

Highly efficient Cas9-mediated transcriptional programming. Chavez A. et. al. (2015) Nature Methods 12, 326–328

State A:

Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.
Jiang, F., Taylor, D.W., Chen, J.S., Kornfeld, J.E., Zhou, K., Thompson, A.J., Nogales, E., Doudna, J.A.(2016) Science 351: 867-871

State B:

Crystal structure of Cas9 in complex with guide RNA and target DNA. Nishimasu, H., Ran, F.A., Hsu, P.D., Konermann, S., Shehata, S.I., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. (2014) Cell(Cambridge,Mass.) 156: 935-949

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 251
  Illegal BglII site found at 1188
  Illegal BamHI site found at 2006
  Illegal XhoI site found at 2962
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2350
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2496
  Illegal BsaI site found at 3158
  Illegal BsaI.rc site found at 1411