Figure 1: Components of the dual-colour live imaging system. | Nature Communications

Figure 1: Components of the dual-colour live imaging system.

From: CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci

Figure 1

(a) A dCas9 protein surrounded by two nuclear localization signals (in yellow) was expressed from a retroviral plasmid containing a puromycin resistance gene. sgRNAs were expressed from a U6 promoter and these could either be transfected or delivered via lentivirus. The sgRNA scaffolds contain double PP7 or MS2 stem loops. The proteins MCP and PCP fused to EGFP or mCherry, respectively, were expressed from lentiviral plasmids. dCas9 allows binding of sgRNAs to their target DNAs and sgRNAs containing MS2 or PP7 stem loops can each recruit four molecules of MCP-EGFP or PCP-mCherry, respectively. (b) Workflow for dual-colour live imaging. To test the system, 3T3 fibroblasts were co-infected by the dCas9 retrovirus and with the MCP-EGFP and PCP-mCherry lentiviruses, followed by puromycin selection and FACS for EGFP and mCherry double positive cells. Single clones based on low background expression of fluorescent proteins and high efficiency of labelling were selected. Optimal clonal cells can then be transduced or transfected with plasmids carrying sgRNAs for subsequent live imaging.

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