Pooled CRISPR Screen in K562 Cells

A previously designed 4 sgRNA/gene CRISPR-Cas9 library was used targeting 5′ ends of conserved exons with sgRNAs varying in length between 19 and 25 base-pairs¹⁴ . The library was generated first by infecting K562 cells with a SFFV-Cas9-BFP vector to create a stably expressed Cas9 cell line. We then infected the lentiviral genome-wide sgRNA library into approximately 120 million cells following the same protocol as the genome-wide shRNA library to maintain at least 1,000-fold representation in cells. Infected cells were selected with puromycin (0.7 μg/mL, Sigma) for 3 days. Percentage of mCherry positive cells was measured by flow cytometry (BD Accuri C6). Selected cells were spun out of selection and into normal RPMI 1640 media. At T0, 120 million cells were spun down (300g for 5 min). Cells were then split into two populations and grown for 14 days, maintaining logarithmic growth (500,000 cells/mL) each day. After 14 days of growth, cells were pelleted by centrifugation, and genomic DNA was extracted for all three time samples separately following Qiagen’s Blood Maxi Kit instructions. sgRNA encoding constructs were analyzed by deep sequencing.