CRISPR/Cas9-mediated mutagenesis of Ago2 and Siwi in silkworm cultured cells.

The BmN-4 cell line, originated from the silkworm Bombyx mori ovary, possesses endogenous small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways. We performed CRISPR/Cas9-mediated genome editing of Ago2 and Siwi, which are the core factors for siRNA and piRNA pathways, respectively, to understand the importance of the two distinct small RNA pathways in this cell line. We found that approximately half of the alleles contained loss-of-function mutations in both Ago2- and Siwi-mutated cells. The mutated cells grew at a slower rate compared to the control cells, strongly suggesting that the siRNA and piRNA pathways are both crucial for the normal growth of BmN-4 cells. The amounts of piRNAs decreased markedly in the Siwi-mutated cells, but global de-repression of transposable elements was not observed. Although the RNA amount of latently infected RNA virus, Bombyx mori macula-like virus (BmLV), increased in both Ago2- and Siwi-mutated cells, the siRNA and piRNA pathways showed a bias toward targeting BmLV genomic and subgenomic RNA, respectively. These results indicate the common, specific, and crucial roles of the two small RNA pathways in B. mori cultured cells.