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Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses 1 – 3 . Here we show that a previously uncharacterized protein encoded by CXorf21— a gene that is associated with systemic lupus erythematosus 4 , 5 —interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease 4 , 6 – 9 . Loss of this type-I-interferon-inducible protein, which we refer to as ‘TLR adaptor interacting with SLC15A4 on the lysosome’ (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF 10 , 11 . The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus 12 – 14 . The interaction between TASL and SLC15A4 links endolysosomal Toll-like receptors to the transcription factor IRF5, providing a mechanistic explanation for the involvement of the complex in systemic lupus erythematosus.

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a “collateral effect” of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.

The class 2 type VI RNA-guided RNA-targeting CRISPR–Cas effector Cas13 can be engineered for RNA knockdown and binding, expanding the CRISPR toolset with a flexible platform for studying RNA in mammalian cells and therapeutic development. A CRISPR way to knockdown RNA CRISPR–Cas prokaryotic defence systems have provided versatile tools for DNA editing. Here, the authors demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR–Cas effector Cas13a (previously known as C2c2) can be engineered for RNA knockdown and binding in mammalian cells. This addition to the CRISPR toolbox expands its potential uses to transcript tracking and knockdown. RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference 1 , 2 , 3 can efficiently knockdown RNAs, but it is prone to off-target effects 4 , and visualizing RNAs typically relies on the introduction of exogenous tags 5 . Here we demonstrate that the class 2 type VI 6 , 7 RNA-guided RNA-targeting CRISPR–Cas effector Cas13a 8 (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli . LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR–Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.

The mutagenic repair of Cas9 generated breaks is thought to predominantly rely on non-homologous end-joining (NHEJ), leading to insertions and deletions within DNA that culminate in gene knock-out (KO). In this study, by taking focused as well as genome-wide approaches, we show that this pathway is dispensable for the repair of such lesions. Genetic ablation of NHEJ is fully compensated for by alternative end joining (alt-EJ), in a POLQ-dependent manner, resulting in a distinct repair signature with larger deletions that may be exploited for large-scale genome editing. Moreover, we show that cells deficient for both NHEJ and alt-EJ were still able to repair CRISPR-mediated DNA double-strand breaks, highlighting how little is yet known about the mechanisms of CRISPR-based genome editing.

Phagocytosis, the process by which cells engulf large particles, plays a vital role in driving tissue clearance and host defense. Its dysregulation is connected to autoimmunity, toxic accumulation of proteins, and increased risks for infections. Despite its importance, we lack full understanding of all molecular components involved in the process. To create a functional map in human cells, we performed a genome-wide CRISPRko FACS screen that identified 716 genes. Mapping those hits to a comprehensive protein–protein interaction network annotated for functional cellular processes allowed retrieval of protein complexes identified multiple times and detection of missing phagocytosis regulators. In addition to known components, such as the Arp2/3 complex, the vacuolar-ATPase-Rag machinery, and the Wave-2 complex, we identified and validated new phagocytosis-relevant functions, including the oligosaccharyltransferase complex (MAGT1/SLC58A1, DDOST, STT3B, and RPN2) and the hypusine pathway (eIF5A, DHPS, and DOHH). Overall, our phagocytosis network comprises elements of cargo uptake, shuffling, and biotransformation through the cell, providing a resource for the identification of potential novel drivers for diseases of the endo-lysosomal system. Our approach of integrating protein–protein interaction offers a broadly applicable way to functionally interpret genome-wide screens.

RNA plays important and diverse roles in biology, but molecular tools to manipulate and measure RNA are limited. For example, RNA interference (RNAi)1-3 can efficiently knockdown RNAs, but it is prone to off-target effects4, and visualizing RNAs typically relies on the introduction of exogenous tags5. Here, we demonstrate that the class 2 type VI6,7 RNA-guided RNA-targeting CRISPR-Cas effector Cas13a8 (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of fifteen orthologs in E. coli, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts. We demonstrate that LwaCas13a is capable of providing comparable levels of knockdown as RNAi, but with dramatically improved specificity. Moreover, catalytically inactive LwaCas13a maintains targeted RNA binding, allowing for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for RNA targeting with wide applicability for studying RNA in mammalian cells.

Phagocytosis, the process of engulfing large particles by cells, is a multilayered biological activity driving tissue clearance and host defense. Dysregulation of phagocytosis is connected to autoimmunity, accumulation of toxic disease proteins, and increased risks for infections. Despite its importance and multiple roles, we lack a full understanding of the cellular machinery involved in executing and regulating the process, including the coordination with other cellular events. To create a functional map in human cells, we performed a reporter- and FACS-based genome-wide CRISPR/Cas9 knock-out screen that identified 716 genes. Mapping the gene hits to a comprehensive protein-protein interaction network annotated for functional cellular processes, allowed to highlight those protein complexes identified multiple times, to identify missing components of the cellular phagocytosis network, and to suggest functional partition among complexes. We validate complexes known to be involved, such as the Arp2/3 complex, the vacuolar-ATPase-Rag machinery, and the Wave-2 complex, as well as processes previously not or only poorly associated with phagocytosis. Among the novel, phagocytosis-relevant cellular functions validated are the oligosaccharyltransferase complex (MAGT1/SLC58A1, DDOST, STT3B, and RPN2) as well as the hypusine pathway (eIF5A, DHPS, and DOHH). Overall, our network of phagocytosis regulators and effectors maps elements of cargo uptake, cargo shuffling and cargo biotransformation through the cell, providing a valuable resource for the identification of potential novel drivers for diseases of the endo-lysosomal system. We further propose that our approach of mining and integrating publicly available protein-protein interaction data with datasets derived from reporter-based genome-wide screens offers a broadly applicable way to functionally map biological processes onto the molecular machinery of the cell. The validation and interpretation of a FACS reporter-based genome-wide CRISPR/Cas9 knock-out screen through protein-protein interaction data yields a comprehensive view of the molecular network regulating and executing phagocytosis in human cells.

CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA- guided endonucleases. Using a computational sequence database mining approach, we identify two Class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 or Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.