Fig. 3: Gene-therapeutic CRISPR knock-in of cytokine transgenes is efficient and specific for top-ranked novel PAM targets. | Nature Communications

Fig. 3: Gene-therapeutic CRISPR knock-in of cytokine transgenes is efficient and specific for top-ranked novel PAM targets.

From: Personalized CRISPR knock-in cytokine gene therapy to remodel the tumor microenvironment and enhance CAR T cell therapy in solid tumors

Fig. 3: Gene-therapeutic CRISPR knock-in of cytokine transgenes is efficient and specific for top-ranked novel PAM targets.

a Circos plots showing the chromosomal distribution of single-nucleotide variants (SNVs, yellow) and novel PAM sites (red) in SK-N-BE2c and SK-N-AS neuroblastoma cell lines. PAMs tested in knock-in experiments are marked with coloured squares. Radar plots visualize annotated features for these PAMs, named after their respective genes. b Schematic of CRISPR/Cas9-mediated transgene knock-in via homology-directed repair (HDR) following ribonucleoprotein (RNP) electroporation. The linear double-stranded (ds) homology-directed repair donor template (HDRT) consists of 5′ and 3′ homologous arms, 400 base pairs (bp) each, a custom EF1α-derived promoter, the cytokine transgene linked by a P2A self-cleaving peptide to a stainable Q8 reporter (CD34 epitope, CD8 transmembrane domain), followed by a stop codon and synthetic poly(A) (sPA) sequence. c PAM-creating mutation allele frequency analysed by Sanger sequencing of selected targets in SK-N-BE2c and SK-N-AS. d Knock-in efficiency of three cytokines (CXCL10, CXCL11, IFNG) at different target sites, measured by Q8 antigen expression by flow cytometry at day 28 post-electroporation. e Correlation of mean knock-in rate (Q8 antigen expression at day 28) with PAM-annotated features, including CRISPR efficiency scores (Doench and Moreno), PAM copy number, and expression of the gene containing the PAM for selected targets. Each dot and n represent the mean value for the selected locus-cell line combinations from (d). Data presentation: c, d Mean ± SD. Each dot represents one biological replicate and exact n values per condition are provided in the Source Data file. Statistical analysis: e Linear regression for curve fitting with R² values and error bands representing the 95% confidence interval followed by two-sided Spearman correlation analysis (exact p = 0.62, 0.82, 0.0022, 0.019). p values: *<0.05, **<0.01; n.s., not significant. Source data are provided as a Source Data file.

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