Cas9-mediated knockout of IGHMBP2 was performed using two sgRNAs (Synthego) to target exon 2 of IGHMBP2 in combination: 5′-CAGAGAGAUGUUCUCCUGCC-3′ and 5′-CUGAAAGAGCUCCAGAGCCG-3′. Ribonucleoprotein (RNP) complexes were prepared by combining 50 pmol of recombinant Cas9 to 50 pmol of each sgRNA and incubating the mixture for 20 min at 37°C. K562 CRISPRi cells (Gilbert et al, 2014 (link)) were nucleofected (SF Cell Line 4D kit; Lonza) with the RNP. Nucleofected clones were isolated by limiting dilution and indel-containing clonal populations were screened by extracting gDNA using Quick Extract (Biosearch Technologies) for PCR amplification using primers targeting up and downstream the Cas9 cut-site: 5′-GGTTGTGGCATTAACTGCCC-3′ and 5′-CCCACATCAATTGTTGGAC-3′. PCR products were separated on a 3% agarose gel at 100 V, and IGHMBP2 gDNA disruption in select clones was further validated via Sanger sequencing with primer 5′-CTTTACGAGGGTACAAGTCACGG-3′ and Western blotting.
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