Towards defining roles for PIERCE1 and C15ORF65 in multiciliated cells through gene editing

Systemic approaches have identified many cilia-associated genes and their function in cilia, but the roles of PIERCE1 and its’ paralogue C15ORF65 in human ciliogenesis has not been studied. This thesis evaluates the similarities between PIERCE1 and C15ORF65 as candidate cilia genes, the capacity of HBEC3-KT cell differentiation in submerged conditions in addition to ALI, the potential of CRISPR-CAS9 gene editing in HBEC3-KT cells, and the ciliary axonemal localisation of PIERCE1 and C15ORF65. Preliminary analysis suggested increased expression of these genes during mucociliary differentiation, and the proteins could localise at the ciliary axoneme. HBEC3-KT cells were shown to able to undergo mucociliary differentiation to generate multiciliated cells and secretory cells in both air-liquid interface (ALI) and under submerged culture conditions. These cells expressed PIERCE1 and C15ORF65 upon mucociliary differentiation. Both genes, alone and in combination, were edited in HBEC3-KT cells using CRISPR-CAS9 technology with FOXJ1-knockout HBEC3-KT cells as a control. Loss of FOXJ1 blocked HBEC3-KT differentiation into multiciliated cells at the ALI or under submerged conditions. Individual PIERCE1-/- or C15ORF65-/- cells were able to differentiate into multiciliated cells, but PIERCE1-/-C15ORF65-/- HBEC3-KT cells failed to differentiate into multiciliated cells at ALI. It is proposed here that loss of both PIERCE1 and C15ORF65 could affect cilia function significantly and hence these genes should be included in genetic tests for PCD.