A novel CRISPR screen allows multiparametric functional analysis of genetic variants to assess pathogenicity, drug response, and mechanistic effects of mutations on disease. NBThighlight
gRNAs were used in the form of crRNA:tracrRNA duplexes purchased from Integrated DNA Technologies and reconstituted in nuclease-free duplex buffer at 10 or 100 μM. For ribonucleoprotein generation, Alt-R SpCas9 Nuclease V3 from Integrated DNA Technologies was used. ssODNs were purchased as unmodified Ultramer DNA oligonucleotides at 100 μM in IDTE, pH 8.0 from Integrated DNA Technologies.
For human colon epithelial organoids, the cassette was delivered as RNP and ssODNs by electroporation using a NEPA21 electroporation device , as describedwith minor modifications. In brief, 24 h before electroporation, the culture medium of proliferative organoids was supplemented with 10 μM Y-27632 , 5 μM CHIR99021 and 1.25% DMSO. On the day of electroporation, organoids were dissociated into clumps containing 5–15 cells by sequential mechanical dissociation in PBS + 0.
For all assays, unless otherwise indicated, culture medium was changed every three days and cells were split to ~20% confluency, when 70–80% confluency was reached. After cell splitting in PIK3CA/PTEN experiments under starvation culture conditions, cells were replated in complete medium to allow cell attachment and the following day washed with phosphate-buffered saline and then incubated in starvation culture medium. At indicated time points, cells were collected for variant:WT′ analysis.
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