Pooled in vitro and in vivo CRISPR-Cas9 screening identifies tumor suppressors in human colon organoids
UMI validation screen, original data from in vivo tumor suppressor CRISPR/Cas9 screening experiment. Figure 6. Experimental setup: 281 tumor suppressor gRNAs, positive controls, neutral controls and gRNAs targeting essential genes were introduced into the CRISPR-UMI lentivirus (Michlits et al., 2017). Transduced AK organoids (APC-KO and KRASG12D) were transplanted subcutaneously in NSG mice followed by barcode sequencing of the tumor pool (from 10 mice) and 5 individual tumors. Clone and read number were determined by next generation sequencing. The conventional gRNA enrichment analysis was compared to UMI-CRISPR-screening with outlier removal.
Steps to reproduce
NGS results were analyzed using the python script ‘IncidenceAbundance.py’ (Michlits et al., 2017), where the filter for the minimal clone size was removed and a hamming distance of 1 was applied. The reads were collapsed from all UMIs either from all clones (‘conventional analysis’) or after subtraction of the 3 biggest clones per gRNA (‘outlier corrected’).