It is important that all stages are carried out using the stably expressing Cas9 version of each cancer organoid line.
Ensure the cell suspension is mixed thoroughly at all stages to create an even single cell suspension before plating.
Avoid maintenance of organoids in a single-cell state for prolonged periods to retain viability.
Avoid freeze/thaw cycles of lentivirus stocks. Prioritising freshly prepared virus stocks will ensure viral integrity is maintained.
It is advised to use black 96-well plates in this protocol, as luminescence can carry over into surrounding wells in clear plates.
All steps involved in the plate set up, including seeding cells, media, antibiotics and CellTiter-Glo should be carried out using reservoirs and multi-channel pipettes where possible to reduce ergonomic strain and to maintain homogeneous solutions throughout.
The volumes of library titrated may differ depending on the batch of lentivirus or library used. To achieve a 30% MOI there may therefore be a requirement to dilute the virus prior to titrating. For this reason, an increased number of titration points may also be required. This may require some R&D to determine optimal volumes to titrate.
gRNA library transduction and screen;
The gRNA library transduction is carried out in triplicate with 12.5 x10^6 cells seeded per replicate. Expansion of the line to a minimum of 40 x10^6 cells is required for this process.
Following selection at Day 6 the organoid suspension should remain in puromycin selection media for the remainder of the screen.
Unless otherwise stated, all steps should be performed under sterile conditions in a microbiological safety cabinet.