Mentors: Daichi Shonai, Scott Soderling, PhD, Cell Biology
Interrogation of endogenous proteins is pivotal to the study of cell types and disease etiology. CRISPR-Cas9-based approaches allow the insertion of DNA sequences into genes of interest (GOI), providing an opportunity to label endogenous proteins and thus advance proteomics. One such CRISPR-based method is homology-independent universal genome engineering (HiUGE), which utilizes adeno-associated virus (AAV) vectors to deliver both the payload lacking gene-specific sequences and the gene-specific gRNA that integrates the payload into the GOI. While universally compatible with genome targets accessible by CRISPR-Cas9, the question remains of whether a double- versus single-oriented payload or HiUGE donor might affect the target efficiency of this strategy. As such, we transfected NIH3T3-Cas9 cells with different HiUGE donors, conducted immunofluorescence (IF) staining to enable epitope tagging, and quantified tag expression to test the hypothesis that double-oriented HiUGE donors would demonstrate greater or even double the efficiency of their single-oriented counterparts. As we continue comparison between single-oriented HA and double-oriented HA-HA, KI D620N/WT VPS35 and KI WT/WT VPS35, and UltraID and Dual-UltraID, preliminary results show that although the difference across constructs varies, the dual-oriented donors not only successfully integrated despite larger size, but they also correspond to increased expression of tagged endogenous proteins.
