Protocol for performing pooled CRISPR-Cas9 loss-of-function screens

Summary: Phenotypic screens involving pooled CRISPR-Cas9 libraries offer a powerful, rapid yet affordable approach to evaluate gene functions on a global scale. Here, we present a protocol for performing pooled CRISPR-Cas9 loss-of-function screens to identify genetic modifiers using either fluorescence-based or cell death phenotypic readouts. We describe steps for designing and amplifying the library and generating and screening cells. We then detail deep sequencing and statistical analysis using cas9 High Throughput maximum Likelihood Estimator.For complete details on the use and execution of this protocol, please refer to Bersuker et al. (2019),1 Li et al. (2022),2 and Roberts et al. (2022).3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

An Amish founder mutation disrupts a PI(3)P-WHAMM-Arp2/3 complex-driven autophagosomal remodeling pathway.

Actin nucleation factors function to organize, shape, and move membrane-bound organelles, yet they remain poorly defined in relation to disease. Galloway-Mowat syndrome (GMS) is an inherited disorder characterized by microcephaly and nephrosis resulting from mutations in the WDR73 gene. This core clinical phenotype appears frequently in the Amish, where virtually all affected individuals harbor homozygous founder mutations in WDR73 as well as the closely linked WHAMM gene, which encodes a nucleation factor. Here we show that patient cells with both mutations exhibit cytoskeletal irregularities and severe defects in autophagy. Reintroduction of wild-type WHAMM restored autophagosomal biogenesis to patient cells, while inactivation of WHAMM in healthy cell lines inhibited lipidation of the autophagosomal protein LC3 and clearance of ubiquitinated protein aggregates. Normal WHAMM function involved binding to the phospholipid PI(3)P and promoting actin nucleation at nascent autophagosomes. These results reveal a cytoskeletal pathway controlling autophagosomal remodeling and illustrate several molecular processes that are perturbed in Amish GMS patients