Shortening the half-life of genetic engineering tools such as dCas9-VP64 can become a pivotal component to regulate many cellular networks, allowing periodic gene expression and protein levels, thereby governing vital cellular processes. However, currently this effector protein has a prolonged half-life in cells and can cause adverse effects by binding to other regions or interacting with other transcriptional or translational machinery.  This thesis aims to explore the N-End Rule as a novel approach to achieve shorter half-life dCas9-VP64, offering tighter control for future applications in synthetic circuits. 
The need for shorter half-life gene effectors arises from the cytotoxic effects observed with persistence exposure that can lead to off-target effects and unintended modifications, hampering their accuracy and reliability in genetic manipulation. The introduction of short-lived effector proteins also holds significant promise for enhancing temporal control and predictable timing of protein degradation.
The central hypothesis driving this research is that the N-End Rule can be harnessed to shorten the half-life of the artificial transcriptional activator dCas9-VP64 (Varshavsky, 1998). Through site-directed mutagenesis, four amino acids were introduced at the N-terminus of dCas9-VP64, including alanine, valine, serine, and cysteine. These amino acid residues were identified as destabilizing when positioned at N-termini based on the N-acetylation N-End Rule (Nguyen, K.T. et al., 2018).
Experimental design involved genetic construct development, fusion with a fluorescent protein tag for visualization, and validation of N-terminus mutations. Using cycloheximide, a protein synthesis and translation inhibitor, the steady-state level of the dCas9-VP64-eGFP protein was monitored through eGFP fluorescence from a microplate reader assay and through western blot analysis. We observed different phenotypes during induction and tracking of fusion proteins' signals. Protein half-life was measured using the first order rate kinetic equation for protein degradation.
The results revealed the differential half-life of dCas9-VP64 variants, showcasing the potential of the N-End Rule in achieving shorter half-life. The cysteine variant demonstrated the shortest half-life of 37 minutes, indicating its potential suitability for synthetic circuits. dCas9-VP64-eGFP with the wild-type N-terminus exhibited a half-life of 57 minutes, while serine and alanine variants displayed approximately 54 and 61 minutes, respectively. 
In conclusion, this thesis contributes to the advancement of strategies for shortening the half-life of proteins. The implementation of the N-End Rule to achieve this goal exemplifies its potential in optimizing genetic behavior control in future applications with synthetic circuits. By harnessing the N-End Rule, researchers can pave the way for future innovations and advancements in the realm of genetic engineering and molecular biology.