PRJNA676146
Whole genome sequencing of single RPE-1 cells with Cas9-induced chromosomal instability
We used "Look-Seq," a procedure combining long-term live-cell imaging with single-cell whole-genome sequencing of the imaged cells. CRISPR-MN were generated by either Cas9/gRNA RNP lipofection or by the doxycycline-induction system. Because CRISPR genome editing can be limited by p53 induction, we transiently blocked p53 expression by siRNA-mediated knockdown. Micronucleated cells were allowed to divide during continuous fluorescence imaging and the daughter cells were then isolated for whole-genome amplification and single-cell sequencing using a robotically controlled microcapillary. We sequenced 18 daughter pairs derived from micronucleated cells that arose after gene editing with three different guides, including a chr2p guide targeting the erythroid-enhancer of BCL11A.