Team:NTU SINGAPORE/Improve
Collaboration
In our attempt to improve our truncations, we explored 2 possible avenues of improvements. First, we considered if further truncations is possible, by looking at additional combinations and novel truncations. Second, we considered alternative functionalities for our truncated dCas9.
Further Truncations
To push the limits of truncations possible, ∆REC1-1 and ∆REC1-3 was added to our ∆3ple. ∆REC1-1 and ∆REC1-3 were chose as their individual truncations results in near WT gene activation. However, part attempt to combine them with HNH leads to drastic decrease in gene activation. Thus, it is no surprise that when ∆REC1-1 and ∆REC1-3 is combined with ∆3ple, gene activation dropped beyond zero. Curiously, these truncations resulted in MFI below negative control. Further experiments would be required to explore various hypothesis, such as potential CRISPRi activity.
Literature update: REC3 truncation
Janice et al (2017) reported that REC3 domain, a part of the REC1 domain, is responsible for HNH activation with on-target DNA binding. In addition, it was reported that REC3 truncation leads to 1000-fold decrease in cleavage rate, but does not affect DNA binding affinity. Considering that the REC 3 domain has essentially no function in the nuclease null dCas9, and is likely to have minimal impact on binding activity of our dCas9-VPR, the REC 3 truncation was replicated and characterized.
