A CRISPR knockout screen revealed modulators of Chlamydia trachomatis parasitophorous vacuole stability
We transduced HeLa cells, a human cervical epithelial cell line, with the Brunello sgRNA library (which targets 19,114 genes and comprises a total of 77,441 sgRNAs, including about four sgRNAs per gene and 1000 non-targeting control sgRNAs). An aliquot of the transduced cells was collected to determine the composition of the pre-selection cell population (= sample “Pre”). In the selection procedure, we infected transduced cells with C. trachomatis L2/434/Bu, either wildtype (CTL2) or a strain carrying an insertional disruption of cpoS (CTL2-cpoS::cat). Later, we collected cells resistant to infection-mediated killing, that is, cells resistant to late-stage lytic death in the case of CTL2 or premature death in the case of CTL2-cpoS::cat. Hence, we included four distinct conditions: uninfected cells and cells infected with CTL2-cpoS::cat collected at 30 hours post infection (samples “UI30h” and “KO30h”), and uninfected cells and cells infected with CTL2 collected at 60 hours post infection (samples “UI60h” and “WT60h”).  For cell collection, the cells were washed twice with Dulbecco's phosphate-buffered saline (DPBS) in the flasks (to remove dead cells and cell debris), harvested, counted, washed once more with DPBS, pelleted (800 x g, 10 min), and stored at -80°C. Genomic DNA was isolated from all cell pellets using the QIAamp DNA Blood Maxi Kit (Qiagen). Prior to following the manufacturer's instructions, RNase A (800 µg/10 million cells) was added to all samples. DNA was quantified using Qubit dsDNA BR Assay Kit (Thermo-Fisher-Scientific). Guide and unique molecular identifier (UMI) sequences were amplified by PCR using modified primers PCR2_FW (5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACAC-3') and PCR3_fw (5'-AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACG CTCT-3'), respectively. 