CRISPR/Cas9-mediated generation of CD47, QPCTL knockout cells

To generate CD47- and QPCTL-knockout HAP1 cells, cells were co-transfected with PX330 vector encoding sgRNA targeting the QPCTL or CD47 gene and a plasmid containing an expression cassette for a guide RNA targeting the zebrafish TIA gene (5’-GGTATGTCGGGAACCTCTCC-3’ (SEQ ID NO:5)) followed by a CMV promotor sequence driving expression of a blasticidin resistance gene flanked by two TIA target sites ⁴⁶. Co-transfection of these plasmids occasionally results in the incorporation of the blasticidin resistance cassette at the site of the targeted genomic locus by non-homologous end joining, rendering cells resistant to blasticidin while also providing a genomic tag at the site of mutation. Four days after DNA transfection, culture medium was supplemented with 20 μg/mL blasticidin (Invivogen). Surviving colonies were clonally expanded and their mutations and/or genomic incorporation of the blasticidin resistance gene were verified by PCR and Sanger sequencing.

To generate CD47- and QPCTL-knockout A431, A375, A549, DLD1, RKO and SKBR3 cell lines, cells were transfected with pLentiCRISPR v.2 vector (Addgene 52961) encoding sgRNA targeting the QPCTL or CD47 gene. To generate HSPA13-knockout cells, HAP1 cells were transfected with pLentiCRISPR v.2 vector encoding sgRNA targeting the HSPA13 gene. One day after transfection, culture medium was supplemented with 2 μg/mL puromycin for two days. Single-cell clones were expanded to obtain clonal knockout populations.

To generate bulk CD47- and QPCTL-knockout B16F10 cells, cells were transfected with pLentiCRISPR v.2. vector encoding sgRNA targeting the murine QPCTL or CD47 gene. One day after transfection, culture medium was supplemented with 2 μg/mL puromycin for two days. Selected cells were expanded and sorted on the basis of αmCD47-MIAP301^LOW mSIRPα-Fc^LOW (in case of CD47 knockout) and αmCD47-MIAP301^HIGH mSIRPα-Fc^LOW (in case of QPCTL knockout) to obtain bulk knockout populations.

Her2-expressing Ba/F3 cells were generated by retroviral transduction with human HER2 (pMX-puro-Her2), and positive clones were selected using puromycin. To generate Ba/F3-Her2 CD47 and QPCTL-knockout cells, nucleofection was used to deliver pLentiCRISPR v.2. vectors encoding sgRNA targeting either the murine QPCTL or CD47 gene, together with a plasmid containing a Cas9, blasticidin resistance, and GFP expression cassette. One day after nucleofection, culture medium was supplemented with 2 μg/mL blasticidin for two days. Selected cells were expanded and sorted to obtain bulk knockout populations. Next, single cells were isolated and expanded to obtain clonal knockout populations.

Gene disruption was validated by sequence analysis of the relevant gene locus, by TIDE analysis ⁴⁷ and, in case of CD47, by flow cytometry.