Fig. 4: CRISPR/Cas9 and AAV6 mediated targeted integration of a promoter-less cassette allows transgene expression under the control of the endogenous CX3CR1 promoter in human cell lines.

A Schematic representation of the human CX3CR1 locus, with a zoom into intron 4 and exon 5, containing the coding sequence. Target sites of the tested sgRNAs are shown. B Cutting efficiency was measured as a percentage of INDELs of different sgRNAs delivered as RNP complexes with CRISPR/Cas9 in RPMI 8226 and K562 cells. The TIDE software was used to analyze the spectrum and frequency of INDELs. Mean and individual values ± SD are shown, 3 independent experiments. C Schematic representation of the targeted integrations with AAV6 donor templates carrying homology arms matching the chosen sgRNA cutting sites in the CX3CR1 locus. SFFV.YFP – CX exon, promoterless.YFP_SA – CX exon and promoterless.YFP_SA – CX intron donor templates were respectively designed for sgRNA9 (CX exon) and sgRNA5 (CX intron). D Representative FACS plots of RPMI-8226 and K562 cells nucleofected with CRISPR/Cas9 RNPs with the chosen sgRNAs and transduced with the generated AAV6. GFP expression is indicative of the activity of the promoter (promoterless constructs) or efficiency of targeted integration (SFFV-exon and AAVS1 safe harbor control) E Percentage of GFP assessed by FACS in RPMI-8226 and K562 cells edited with CRISPR/Cas9 + AAV6 vectors in the tested and control conditions. F, G Percentage of targeted alleles assessed by ddPCR on bulk, GFP⁺ and GFP^- sorted RPMI-8226 (F) and K562 (G) edited cells. H CX3CR1 gene expression was evaluated with ddPCR on bulk, GFP⁺ and GFP⁻ sorted RPMI-8226 edited cells. Data shown as the fold on mock. E–H Individual replicates and mean values ± SD are shown, 3 indepentent experiments. Source data are provided as a Source Data file.