Abstract

We have set up a facility for CRISPR screening at Amsterdam UMC. Mostly using the human and mouse Toronto Knock-out (TKO) version 3 libraries, we have performed drug, isogenic and FACS-based screens in cell lines from public resource centers, in patient cell lines established in our department, and cell line models that we constructed. Several screens have now been published. To find genes involved in transcription-coupled nucleotide excision repair (TC-NER), we performed a drug screen with Illudin S, and discovered that the previously uncharacterized gene ELOF1 is a component of the human RNA polymerase II complex and a novel TC-NER gene. We further elucidated the mechanism in a wide range of follow-up experiments, including three drug/isogenic CRISPR screens. To investigate the role of leading strand-oriented alternative PCNA clamp loader DSCC1-RFC in DNA replication, repair, and sister chromatid cohesion (SCC), we screened an isogenic DSCC1ko cell line. The MMS22L-TONSL heterodimer was found to function in a SCC establishment pathway parallel to the DSCC1-RFC complex. To identify novel genes involved in sister chromatid cohesion, we performed isogenic screens in two cell lines displaying cohesion defects, ESCO2mut and DDX11ko. Apart from many genes which were known to play a role in mitotic progression and cohesion, we found PAXIP1 as a top hit. Further validation experiments, including an isogenic PAXIP1ko CRISPR screen, confirmed the PAXIP1-PAGR1 complex as a novel regulator of cohesin occupancy on chromatin. The role in cohesin regulation might clarify previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair. For statistical analysis of isogenic screens, we have made an adapted version of the DrugZ software, IsogenicZ (available on GitHub), that normalizes sgRNA readcounts based on differences in t=0 reads to correct for sampling error at transduction. Other current interests of our group include CRISPR library design, using different Cas proteins for screening, and in vivo screens in mice.