Whole-genome CRISPR Screening of stably expressing Cas9 Cancer Organoid Lines v1

This protocol is for whole-genome CRISPR screening of stably expressing Cas9 cancer organoid lines in triplicate using the commercially available minimal genome-wide human CRISPR Cas9 library. The protocol uses lentiviral transduction as a method for gRNA delivery. This method can be adapted for other gRNA libraries. The protocol can be followed assuming the following is known: - The number of days required for screening - The size of the gRNA library - The required coverage of the library To allow for efficient scale up of the organoid culture for screen, a 5% suspension culture method can be used. This is not essential to be able to perform whole-genome CRISPR screening of stably expressing Cas9 cancer organoid lines, but provides a more scalable, ergonomic and cost efficient culturing method. Prior to commencing the screen a puromycin antibiotic titration is used to identify the most suitable puromycin concentration for the selection of Cas9 positive cancer organoid lines transduced with gRNA library virus. A gRNA library titration is then performed to determine the volume of library virus required to transduce Cas9 cancer cells at 30% transduction efficiency which is calculated using FACS analysis. This is to avoid host cells taking up more than one gRNA copy per cell, therefore an MOI of 30% should be aimed for. Process Diagram This protocol uses a 21-23 day screen process.