Fig. 4: Multiplexing in the CRISPR–dCas9 system.

To simultaneously visualize different genomic regions, multicolor approaches have been applied to the CRISPR–dCas9 system by utilizing dCas9 orthologs from different species or by combining RNA aptamers with their corresponding coat proteins. Top: dCas9 proteins derived from Sp, Nm and St1 were labeled with RFP, GFP and BFP, respectively. By designing sgRNAs to target different genomic regions with distinct PAM requirements, three separate genomic sites were imaged simultaneously. Bottom: alternatively, distinct RNA aptamers (for example, MS2, PP7 and boxB) were incorporated into sgRNAs targeting different genomic regions. These aptamers were paired with their respective coat proteins fused to FPs with separated spectral properties (for example, MCP–BFP, PCP–GFP and N22–RFP), visualizing multiple genomic regions at the same time.