Gecko v2 sgrna library

The human codon-optimized sequence of S. pyogenes Cas9 was subcloned from plasmid lentiCas9-Blast (Addgene #52962) into the pQCXIH retroviral vector (Clontech), which was used to generate retroviruses to transduce HeLa cells. Mixed stable cells were selected in the presence of hygromycin B (200 μg/ml, Life Technologies). Lentivirus sgRNA libraries were generated following published protocols using the human GeCKO v2 sgRNA library (Addgene #1000000049)^{19 (link)}. The GeCKO v2 library is composed of two half-libraries (library A and library B). Each half-library contains three unique sgRNA per gene and was independently screened with toxins. Cells were transduced with lentivirus-packaged sgRNA library at a MOI of 0.2.

Genome-wide CRISPR/Ca9 screen was performed to identify genes essential for T-ALL survival, using KOPT-K1 cell line as the model system. Experimental procedure was based on previously published protocol(52 (link)) with minor modifications. Briefly, Cas9-expressing KOPT-K1 cells were first generated by lentiviral transduction, and single cell dilution was performed to develop and select clones with high level of Cas9 for genome-wide CRISPR screen. Cas9-expressing KOPT-K1 cells were transfected with the GeCKO V2 sgRNA library (Addgene, #1000000048) at low multiplicity of infection (0.3). Transfected cells were selected with puromycin for two days and then cultured for another 6 days to allow proliferation. Genomic DNA was extracted from surviving cells and sgRNAs were amplified by PCR followed by Illumina sequencing(53 ). The sgRNA enrichment was quantified based on read counts, and fold change from baseline (day 0) to day 6 was used for estimate gene dependency score(53 ).