Noell, G. | Search bioRxiv

bioRxiv2022

Optimised whole-genome CRISPR interference screens identify ARID1A-dependent growth regulators in human induced pluripotent stem cells

Perturbation of gene function is a powerful way to understand the role of individual genes in cellular systems. Whole-genome CRISPR/Cas-based screens have parallelized this approach and identified genes that modulate growth in many contexts. However, the DNA break-induced stress upon Cas9 action limits the efficacy of these screens in important models, such as human induced pluripotent stem cells (iPSCs). Silencing with a catalytically inactive Cas9 is a less stressful alternative, but has been considered less effective so far. Here, we first tested the efficiency of several dCas9 fusion proteins for target repression in human iPSCs, and identified dCas9-KRAB-MeCP2 as the most potent. We then produced monoclonal and polyclonal cell lines carrying this construct from multiple iPSC donors, and optimized genome-wide screens with them. We found silencing in a 200bp window around the transcription start site to be as effective as using wild-type Cas9 for identifying essential genes in iPSCs, but with a reduced cost due to better cell survival. Monoclonal lines performed better, but data from polyclonal lines were of sufficient quality for screening for larger effects. Finally, we performed whole-genome screens to identify dosage sensitivities that depend on the functionality of ARID1A, a commonly mutated cancer and developmental disorder gene. We observed differential growth upon depletion of NF2, TAF6L, EZH2, and PSMB2 genes in ARID1A+/-lines compared to wild type, and an enrichment of proteasome genes. Further, we confirmed that the context-specific growth decrease was phenocopied by treating the cells with a proteasome inhibitor, suggesting a pharmacologically targetable synthetic lethal interaction between the proteasome and ARID1A. We propose that many more plausible targets in challenging cell models can be efficiently identified with our approach.

cell biology↗

bioRxiv2019

Minimized double guide RNA libraries enable scale-limited CRISPR/Cas9 screens

Genetic screens based on CRISPR/Cas technology are a powerful tool for understanding cellular phenotypes. However, the coverage and replicate requirements result in large experiment sizes, which are limiting when samples are scarce, or the protocols are expensive and laborious. Here, we present an approach to reduce the scale of genome-wide perturbation screens up to fivefold without sacrificing performance. To do so, we deliver two randomly paired gRNAs into each cell, and rely on recent advances in gRNA design, as well as availability of gRNA effect measurements, to reduce the number of gRNAs per gene. We designed a human genome-wide library that has effective size of 30,000 constructs, yet targets each gene with three gRNAs. Our minimized double guide RNA library gives similar results to a standard single gRNA one, but using substantially fewer cells. We demonstrate that genome-wide screens can be optimized in a demanding model of induced pluripotent stem cells, reducing reagent cost 70% per replicate compared to conventional approach, while retaining high performance. The screen design and the reduction in scale it provides will enable functional genomics experiments across many possible combinations of environments and genetic backgrounds, as well as in hard to obtain and culture primary cells.

genetics↗