School of Science Division of Life Science 26 CRISPR/Cas9 Analysis of Essential Genes Supervisor: POON Randy Yat Choi / LIFS Student: LIN Muyun / BCB Course: UROP1100, Summer In vivo gene knockout, like RNA interference and the CRISPR/Cas-9 system are robust and powerful tools in research studies and have been proved to have a wide variety of applications. Here, I tested the effectiveness of a new technique involving nanobodies which aims at degrading proteins post-transcriptionally via the highly conserved ubiquitin pathway. As the exploratory stage of investigating the functions of nanobodies, starting with the relatively well-studied protein, GFP (Green Fluorescence Protein), will stand as an excellent example for later studies. In total, three different nanobodies were tested: the NSlmb-vhhGFP4, AIDvhhGFP4 and SPOP-vhhGFP4. Preliminary results showed that the nanobodies worked in degrading H2B-GFP in a considerable effectiveness. Cloning and Characterization of Novel Cytoskeletal Regulators Supervisor: QI Robert Zhong / LIFS Student: CATIELLO Carlo / BIOT Course: UROP1100, Fall UROP2100, Spring Microtubules are major components of the cytoskeleton which are formed by α and β tubulins and are essential for the organization of eukaryotic cells, playing an important part in vital cellular processes such as intracellular transport, structural support, and cell division. The initiation of microtubule growth, microtubule nucleation, occurs at the microtubule-organizing centers (MTOCs) and is regulated by the γtubulin ring complex (γTuRC), which mainly consists of γ-tubulin and γ-tubulin complex proteins (GCPs). From the previous experiment, it was found that a phosphomimetic mutant of γ-tubulin, γ-tubulin-GFP-Twinstrep S364D have binding defects to GCP proteins, making them harder to be incorporated into y-TuRC. We will conduct experiments, RNA interference knockdown as well as microtubule regrowth assay to further study how mutations affect nucleation in centrosomes.