CRISPR Targeting of ATRX Exon 9

The ATRX cDNA was screened by ZiFiT to determine a CRISPR target in exon 9 of ATRX [37 (link)]. Two complementary oligos were designed by ZiFit, ATRXex9_1 and ATRXex9_2 (Supplementary Table 1), and were subsequently annealed and ligated into a CRISPR RNA expression plasmid (MLM3636; Addgene). The resulting plasmid was co-transfected with a Cas9 nuclease expression plasmid (41815; Addgene) into wild-type HCT116. Cells were subcloned, and screened for correct targeting by interrogating the disruption of an SmlI restriction enzyme site that lies directly adjacent to the target sequence cut by the Cas9 endonuclease. Targeting PCR was performed using ATRXex9F and ATRXex9R (Supplementary Table 1), and products were subjected to SmlI digestion. Sanger sequencing of the correctly modified clones was performed with ATRXex9SeqF to confirm that an early stop codon was inserted or a frame-shift mutation had occurred.