Analysis Tools
cellular
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• after introduction of single guide RNAs (sgRNAs) targeting exonic Tmem258 sequences (Tmem258 CRISPR) by transduction with lentiviral particles co-encoding Cre recombinase, spheroid cultures derived from colonic crypts exhibit fewer viable Cas9-GFP-positive cells than colonic organoid cultures that have received control sgRNAs targeting noncoding intronic Tmem258 regions
• morphologically, Tmem258 CRISPR organoids contain scattered Cas9-GFP-positive cells within small spheroids that encapsulate numerous dead cells within the lumen, whereas control CRISPR organoids appear healthier and larger with contiguous Cas9-GFP-positive cells suggestive of active proliferation
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• after simultaneous delivery of Cre recombinase and sgRNA targeting of Tmem258 sequences by lentiviral transduction, Tmem258 CRISPR organoid cultures contain more in-frame mutations in Cas9-GFP-positive cells than control CRISPR organoids, suggesting that out-of-frame alleles are negatively selected, consistent with increased ER stress
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