Cas9 Stable Cell Line - HCT116

The researchers used an auxin-inducible degradation system to create a Cas9 stable cell line, the HCT116 cell line, that can quickly eliminate ORC2, revealing a variety of cell division cycle phenotypes. First, the researchers found that each subunit of ORC and CDC6 is essential in human cells through tiling-sgRNA CRISPR screening. Then, the researchers observed in these stable cell lines that in the absence of ORC2, the cells' ability to load MCM2-7 on G1 phase chromatin was weakened, resulting in difficulties in initiating DNA replication or the inability of the cell division cycle to proceed normally. In addition, ORC2-deficient cells have larger nuclei and decompressed heterochromatin. Some ORC2-deficient cells that completed DNA replication entered mitosis but never exited. ORC1 knockout cells also exhibited extremely slow cell proliferation rates and abnormal cell and nuclear morphology. Therefore, ORC proteins and CDC6 are essential for normal cell proliferation and nuclear organization.

Figure 1. Using tilling-sgRNA CRISPR screening, the researchers found that each subunit of ORC and CDC6 is essential in human cells. Through the auxin-induced degradation system, they created a Cas9 stable cell line that can rapidly eliminate ORC2, revealing a variety of cell division cycle phenotypes. The main defect of ORC2 deficiency is that cells encounter difficulties in initiating DNA replication or blocked cell division cycle progression in the G1 phase, resulting in reduced loading of MCM2-7 on chromatin. (Chou HC, et al., 2021)