Lentiviral CRISPR-Cas9 Targeting Bcl-xL

We obtained a pRRL plasmid expressing a Cas9-T2A cassette from Daniel Stetson (University of Washington), described in Gray et al., 2016 [55 (link)]. Guide RNAs specific to each gene were inserted into pRRL using the In-Fusion cloning system (Takara Bio). Briefly, sense and anti-sense sgRNAs were annealed to form a gRNA. These gRNAs were cloned into the pRRL plasmid using the In-Fusion enzyme (Takara Bio). Cloned plasmids were then transformed into Stellar Competent E. Coli cells and antibiotic selected. Single colonies were grown up and plasmids were isolated using the Qiagen Mini-Prep Kit. Lentivirus targeting Bcl-xL or the non-targeting control was generated by co-transfection of 293T cells.