Part:BBa_K2818000

∆RuvCIII-2 ∆HNH ∆REC2 4QC_HNHpK Sp-dCas9

dCas9 is a catalytically inactive Cas9, which still retains it's ability to bind to DNA. This truncated Sp-dCas9 has been improved upon to increase DNA binding affinity. It can be fused to an effector protein to achieve targetting to specific locus. This is an improvement from part Part:BBa_K2316001

Usage and Biology

It was previously shown that SpdCas9 can be truncated and improved by mutation of DNA backbone proximal amino acids. This part is a further improvement upon Part:BBa_K2316001, where additional mutation and site specific fusion of a cationic peptide demonstrated further improvement upon the truncated dCas9.

Improving upon previously submitted part ∆RuvCIII-2 ∆HNH ∆REC2 Sp-dCas9 Enhanced (BBa_K2316001), also referred to as ∆3ple 5_6_10, mutation at the position 15 is found to produce the best performing variant. Further compounding of mutations appear to no improve ∆3ple 5_6_10_15. These mutations are indicated in short form on name of this part as “4QC”.

Figure 1. Further compounding of mutations upon BBa_K2316001 (3ple_5_6_10).

In addition, small cationic DNA binding peptide “pK” is fused to the HNH position. This position is found to be the best performing, compared to N-terminus and Rec2 position fusion. This is especially so in endogenous gene activation results as seen in Figure 3 where ∆3ple 5_6_10_15 HNH-pK demonstrates consistently higher gene expression.

Figure 2. Reporter gene activation by pK fused to different positions on ∆3ple 5_6_10_15.

Figure 3. Endogenous gene activation by pK fused to different positions on ∆3ple 5_6_10_15.

The performance of 3ple 5_6_10_15 HNH-pK was demonstrated in the context of transcriptional activator VPR fusion for expression upregulation of the tested genes, as described by Chavez et. al. (2015).

This part only contains the ∆3ple 5_6_10_15 HNH-pK tCas9 variant, and is not fused to any effector proteins.

References

Chavez, A., Scheiman, J., Vora, S., Pruitt, B. W., Tuttle, M., P R Iyer, E., … Church, G. M. (2015). Highly efficient Cas9-mediated transcriptional programming. Nat Methods, 12(4), 326–328.

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 804
  Illegal BamHI site found at 1622
  Illegal XhoI site found at 2608
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1996
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2142
  Illegal BsaI site found at 2804
  Illegal BsaI.rc site found at 1027