Part:BBa_K2316001
∆RuvCIII-2 ∆HNH ∆REC2 Sp-dCas9 Enhanced
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, Rec2 domain and RuvCIII-2 domain of the SP-dCas9. The triple truncation (∆REC2 ∆RuvCIII-2 ∆HNH, referred to as ‘∆3ple’) is especially of interest, since it reduces the size of the dCas9 gene to around 3.2kB which is on par with the size of Sa-Cas9. The ∆3ple truncation has slightly lower activation compared to double truncation. In order to improve the activity of ∆3ple dCas9, quick changes are further performed at various site of dCas9. Combination of quick change at Asn497Lys (QC5), Thr657Lys (QC6) and Ser1109Arg (QC15) show improved activity. It's binding capability was tested by assessing the strength in transcriptional activation via by tagging it with a VP64-p65-Rta tripartite activator.
Activation of a zsGreen reporter gene using the various truncations.
Quick change mutations done on ∆3ple. QC5 and QC15 appears to give best improvement in gene activation.
Combination of different mutations done on ∆3ple QC5 and QC15 as base. QC5+6 and QC15+6 appears to give best improvement in gene activation.
Combination of different mutations done on ∆3ple QC5+6 and QC15+6 as base.
Sequence and Features| None |