CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), Cas9 can be reprogrammed to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently-devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.

LC3-associated phagocytosis (LAP) is a process wherein elements of canonical autophagy conjugate LC3 to the membranes of phagosomes, facilitating maturation upon fusion to lysosomes. Here, we characterize the molecular requirements for LAP, and identify a protein, Rubicon, required for LAP but not canonical autophagy. During LAP, Rubicon is recruited to the LAP-engaged phagosome (“LAPosome”) and is required for the activity of a Class III PI3K complex containing Beclin-1, UVRAG, and VPS34, but lacking the canonical autophagy components ATG14 and Ambra1. This allows for the sustained localization of PI(3)P, which is critical for the recruitment of downstream autophagic proteins and the stabilization of the NOX2 complex to produce ROS. Both PI(3)P and ROS, as well as ATG7, ATG3, ATG5, ATG12, and ATG16L, are required for the conjugation of LC3 to the LAPosome and subsequent association with LAMP1+ lysosomes. LAP is also induced by engulfment of Aspergillus fumigatus, a fungal pathogen that commonly afflicts immunocompromised hosts, and is required for its optimal clearance in vivo. Therefore, we have identified molecules that distinguish LAP from canonical autophagy, thereby elucidating the importance of LAP in the response to Aspergillus fumigatus infection.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here, we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, MERS-CoV, bat coronavirus HKU5 expressing the SARS-CoV-1 spike, and VSV expressing the SARS-CoV-2 spike. We identify known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways including HMGB1 and the SWI/SNF chromatin remodeling complex that are SARS-lineage and pan-coronavirus specific, respectively. We show HMGB1 regulates ACE2 expression and is critical for viral entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. Together this identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS-lineage specific and pan-coronavirus host factors that regulate susceptibility to highly pathogenic coronaviruses.

Defects in dying cell clearance are postulated to underlie the pathogenesis of systemic lupus erythematosus (SLE)1. Mice lacking molecules associated with dying cell clearance develop SLE-like disease2, and phagocytes from SLE patients often display defective clearance and increased inflammatory cytokine production when exposed to dying cells in vitro. Previously, we3–6 and others7 described a form of noncanonical autophagy called “LC3-associated phagocytosis” (LAP), wherein phagosomes containing engulfed particles, including dying cells3,4,7, recruit elements of the autophagy pathway to facilitate phagosome maturation and digestion of cargo. Genome-wide association studies have identified polymorphisms in atg58 and possibly atg79, involved in both canonical autophagy and LAP3–7, as predisposition markers for SLE. Here, we describe the consequences of defective LAP in vivo. Mice lacking any of several components of the LAP pathway display elevated serum inflammatory cytokines, autoantibodies, glomerular immune complex deposition, and evidence of kidney damage. Dying cells, injected into LAP-deficient animals, are engulfed but not efficiently degraded, and trigger acute elevation of pro-inflammatory cytokines but not the anti-inflammatory interleukin (IL)-10. Repeated injection of dying cells into LAP-deficient, but not LAP-sufficient animals accelerated SLE-like disease, including increased serum levels of autoantibodies. In contrast, animals deficient for genes required for canonical autophagy but not LAP do not display defective dead cell clearance, inflammatory cytokine production, or SLE-like disease, and like wild-type animals, produce IL-10 in response to dying cells. Therefore, defects in LAP, rather than canonical autophagy, can cause SLE-like phenomena, and may contribute to the pathogenesis of SLE.

Human noroviruses (NoV) are a leading cause of gastroenteritis globally, yet host factors required for NoV infection are poorly understood. We identified host molecules essential for murine NoV (MNoV) induced cell death including CD300lf as a proteinaceous receptor. CD300lf is essential for MNoV binding and replication in cell lines and primary cells. Additionally, Cd300lf−/− mice are resistant to MNoV infection. Expression of CD300lf in human cells breaks the species barrier restricting MNoV replication. The crystal structure of the CD300lf ectodomain revealed a potential ligand binding cleft composed of residues critical for MNoV infection. Therefore, the presence of a proteinaceous receptor is the primary determinant of MNoV species tropism while other components of cellular machinery required for NoV replication are conserved between humans and mice.