Multiple chromosomal sites are readily labeled using Cas9 and guide RNAs that bind fluorescent proteins, enabling visualization of chromatin dynamics. A lack of techniques to image multiple genomic loci in living cells has limited our ability to investigate chromosome dynamics. Here we describe CRISPRainbow, a system for labeling DNA in living cells based on nuclease-dead (d) Cas9 combined with engineered single guide RNA (sgRNA) scaffolds that bind sets of fluorescent proteins. We demonstrate simultaneous imaging of up to six chromosomal loci in individual live cells and document large differences in the dynamic properties of different chromosomal loci.

Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow