Pooled in vitro and in vivo CRISPR-Cas9 screening identifies tumor suppressors in human colon organoids

Published: 20 April 2020| Version 1 | DOI: 10.17632/sxrvzkr4hh.1
Contributor:
Henner Farin

Description

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.

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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’).

Institutions

Deutsches Krebsforschungszentrum

Categories

Cell Biology

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