Difference between revisions of "Part:BBa K4727008"

Revision as of 13:50, 1 October 2023

dCas9 standardized

Dead Cas9 (dCas9) is the modified version of the Cas9 enzyme, in which the two nuclease domains, HNH and RuvC, are catalytically inactive. Therefore, the protein is unable to cleave the DNA but can only bind to the target DNA sequence that is complementary to the guide RNA [1]. The dCas9:sgRNA complex performs the transcription repression or the modulation of the expression of the target.

The coding sequence for dCas9, registered as part <partname> BBa_J107202 </partname>, though, is not suitable as a BioBrick RFC[10] standard part due to the presence of an EcoRI site in position 1339 5'-end of the dCas9 coding sequence. This new registered part solves this problem, as the EcoRI restriction site present in the sequence, has been removed and mutagenised altering the sequence with a silent mutation that maintains the codon usage in E. coli unvaried.

Usage and Biology

Assessing silencing efficiency

Once introduced the silence mutation the the coding sequence of the dCas9 we wanted to assess if any significant alteration was measurable in the silencing efficacy of the dCas9 coded by the mutated sequence. For this a plate reader experiment was set up with the following specifications. Escherichia coli TOP10 chemically competent cells were co-transformed with three plasmids bearing:0 (a) BBa_I13521 expression cassette, (b) HSL inducible pTET guide RNA, (c) dCas expression cassette. Two different transformations were made, with the only difference of the dCas construct: in one case, the newly mutated and in the other the one of part BBa_J107202. Different HSL inductions of the pTET guide have been provided to demonstrate silencing efficacy

From this analysis it is possible to infer that no significant difference can be seen in the silencing efficacy of the mutated sequence of the dCas9. Overall both the samples do not reach complete silencing of the RFP expression levels probably due to the high copy number of the plasmid bearing the I13521 expression cassette. At the same time, even at no levels of HSL induction (0) the expression of RFP i lower than the reference level, this can be explained by the leakage activity of the pLux inducible promoter.

Sequence and Features

References

[1] Mahas, Ahmed, C. Neal Stewart, e Magdy M. Mahfouz. «Harnessing CRISPR/Cas Systems for Programmable Transcriptional and Post-Transcriptional Regulation». Biotechnology Advances 36, fasc. 1 (gennaio 2018): 295–310. https://doi.org/10.1016/j.biotechadv.2017.11.008.