Difference between revisions of "Part:BBa K2361000"

Revision as of 19:05, 31 October 2017

spdCas9

This part contains the protein coding sequence for the dCas9 originating from Streptococcus pyogenes. This protein is a catalytically-dead Cas9 variant, which lacks endonuclease activity. It is used for gene repression using CRISPR-interference. It can been seen as an improvment of part BBa_K1026001 since the illegal EcoRI site has been removed and the part was fully sequenced (see experience).

Usage and Biology

Overexpression of dCas9 may cause cytotoxic effects [1], therefore we recommend expressing it using an inducible promotor. In this part the start codon (ATG) starts directly behind the XbaI site and not at the last A of the XbaI site. This may lead to reduced efficiency of expression when combined with RBS's from the iGEM repository, but more importantly this needs to be taken into consideration when creating fusion proteins with this part to ensure that no frame-shifts occur. The first step of construction was amplifying the part from

References

[1] Nielsen, A. A., & Voigt, C. A. (2014). Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Molecular Systems Biology, 10(11), 763. http://doi.org/10.15252/msb.20145735

Sequence and Features