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Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40–75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.

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Recent case reports and epidemiological data suggest fungal infections represent an under-appreciated complication among people with severe COVID-19. However, the frequency of fungal colonization in patients with COVID-19 and associations with specific immune responses in the airways remain incompletely defined. We previously generated a single-cell RNA-sequencing (scRNA-seq) dataset characterizing the upper respiratory microenvironment during COVID-19, and mapped the relationship between disease severity and the local behavior of nasal epithelial cells and infiltrating immune cells. Our study, in agreement with findings from related human cohorts, demonstrated that a profound deficiency in host immunity, particularly in type I and type III interferon signaling in the upper respiratory tract, is associated with rapid progression to severe disease and worse clinical outcomes. We have now performed further analysis of this cohort and identified a subset of participants with severe COVID-19 and concurrent detection ofCandidaspecies-derived transcripts within samples collected from the nasopharynx and trachea. Here, we present the clinical characteristics of these individuals, including confirmatory diagnostic testing demonstrating elevated serum (1, 3)-β-D-glucan and/or confirmed fungal culture of the predicted pathogen. Using matched single-cell transcriptomic profiles of these individuals’ respiratory mucosa, we identify epithelial immune signatures suggestive of IL-17 stimulation and anti-fungal immunity. Further, we observe significant expression of anti-fungal inflammatory cascades in the nasal and tracheal epithelium of all participants who went on to develop severe COVID-19, even among participants without detectable genetic material from fungal pathogens. Together, our data suggests that IL-17 stimulation – in part driven byCandidacolonization – and blunted type I/III interferon signaling represents a common feature of severe COVID-19 infection.

SUMMARYRecent microbial genome sequencing efforts have revealed a vast reservoir of mobile genetic elements containing integrases that could be useful genome engineering tools. Large serine recombinases (LSRs), such as Bxb1 and PhiC31, are bacteriophage-encoded integrases that can facilitate the insertion of phage DNA into bacterial genomes. However, only a few LSRs have been previously characterized and they have limited efficiency in human cells. Here, we developed a systematic computational discovery workflow that identifies thousands of new LSRs and their cognate DNA attachment sites by. We validate this approach via experimental characterization of LSRs in human cells, leading to three classes of LSRs distinguished from one another by their efficiency and specificity. We identify landing pad LSRs that efficiently integrate into synthetically installed attachment sites orthogonal to the human genome, human genome-targeting LSRs with computationally predictable pseudosites, and multi-targeting LSRs that can unidirectionally integrate cargos at with similar efficiency and superior specificity to commonly used transposases. LSRs from each category were functionally characterized in human cells, overall achieving up to 7-fold higher plasmid recombination than Bxb1 and genome insertion efficiencies of 40-70% with cargo sizes over 7 kb. Overall, we establish a paradigm for large-scale discovery of microbial recombinases and reconstruction of their target sites directly from microbial sequencing data. This strategy provides a rich resource of over 60 experimentally characterized LSRs that can function in human cells and thousands of additional candidates for large-payload genome editing without exposed DNA double-stranded breaks.

Transcriptome engineering requires flexible RNA-targeting technologies that can perturb mammalian transcripts in a robust and scalable manner. CRISPR systems that natively target RNA molecules, such as Cas13 enzymes, are enabling rapid progress in the investigation of RNA biology and advancement of RNA therapeutics. Here, we sought to develop a Cas13 platform for high-throughput phenotypic screening and elucidate the design principles underpinning its RNA targeting efficiency. We employed the RfxCas13d (CasRx) system in a positive selection screen by tiling 55 known essential genes with single nucleotide resolution. Leveraging this dataset of over 127,000 guide RNAs, we systematically compared a series of linear regression and machine learning algorithms to train a convolutional neural network (CNN) model that is able to robustly predict guide RNA performance based on guide sequence alone. We further incorporated secondary features including secondary structure, free energy, target site position, and target isoform percent. To evaluate model performance, we conducted orthogonal screens via cell surface protein knockdown. The final CNN model is able to predict highly effective guide RNAs (gRNAs) within each transcript with >90% accuracy in this independent test set. To provide user interpretability, we evaluate feature contributions using both integrated gradients and SHapley Additive exPlanations (SHAP). We identify a specific sequence motif at guide position 15-24 along with selected secondary features to be predictive of highly efficient guides. Taken together, we derive Cas13d guide design rules from large-scale screen data, release a guide design tool (http://RNAtargeting.org) to advance the RNA targeting toolbox, and describe a path for systematic development of deep learning models to predict CRISPR activity.

SARS-CoV-2 infection can cause severe respiratory COVID-19. However, many individuals present with isolated upper respiratory symptoms, suggesting potential to constrain viral pathology to the nasopharynx. Which cells SARS-CoV-2 primarily targets and how infection influences the respiratory epithelium remains incompletely understood. We performed scRNA-seq on nasopharyngeal swabs from 58 healthy and COVID-19 participants. During COVID-19, we observe expansion of secretory, loss of ciliated, and epithelial cell repopulation via deuterosomal cell expansion. In mild and moderate COVID-19, epithelial cells express anti-viral/interferon-responsive genes, while cells in severe COVID-19 have muted anti-viral responses despite equivalent viral loads. SARS-CoV-2 RNA+ host-target cells are highly heterogenous, including developing ciliated, interferon-responsive ciliated, AZGP1high goblet, and KRT13+ “hillock”-like cells, and we identify genes associated with susceptibility, resistance, or infection response. Our study defines protective and detrimental responses to SARS-CoV-2, the direct viral targets of infection, and suggests that failed nasal epithelial anti-viral immunity may underlie and precede severe COVID-19., Graphical abstract, A study of nasopharyngeal swabs from healthy and COVID-19-infected individuals shows how infection leads to compositional changes in the respiratory epithelium, with early dampened antiviral responses in the nasal epithelia likely underlying and preceding severe disease.

Single-cell analysis of CNS interactions Despite their importance in the physiology and pathology of the central nervous system (CNS), few methods are available for the unbiased, systematic investigation of cell-to-cell interactions at single-cell resolution. Clark et al. developed RABID-seq, a method that combines barcoded viral tracing with single-cell RNA sequencing (see the Perspective by Silvin and Ginhoux). RABID-seq identified the axon guidance molecules Sema4D-PlexinB2 and EphrinB3-EphB3 as mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis and, potentially, multiple sclerosis. These studies also identified candidate therapeutic molecules for the modulation of microglia-astrocyte interactions in multiple sclerosis. Science , this issue p. eabf1230 ; see also p. 342

Recent case reports and epidemiological data suggest fungal infections represent an under-appreciated respiratory co-infection among people with severe COVID-19, however the frequency and clinical impact is currently unknown. We have previously generated a single-cell RNA-sequencing (scRNA-seq) dataset characterizing the upper respiratory microenvironment during COVID-19 and mapped the relationship between disease severity with the local behavior of nasal epithelial and immune cells. In agreement with other human cohorts, this prior study suggested that a deficiency in host immunity, particularly in type I/ III interferon signaling in the upper respiratory tract, is associated with rapid progression to severe disease and worse clinical outcomes. Here, we have performed further analysis of this cohort and identified a subset of participants with severe COVID-19 and concurrent nasal and/or endotracheal infection with Candida spp. We present the clinical characteristics of these individuals, including confirmatory testing demonstrating elevated serum (1,3)-β-D-glucan and/or a positive fungal culture. Finally, using the matched single-cell transcriptomic profiles of these individuals’ respiratory mucosa, in addition to blunted type I/III interferon signaling previously reported, we identified epithelial and mucosal immune signatures consistent with augmented IL-17 responses, suggestive of activated anti-fungal immunity. These results indicate that a subset of patients with severe COVID-19 exhibit increased frequency of concurrent fungal infections associated with augmented IL-17 responses and suggest that concurrent fungal infection may be contributing to SARS-C0V-2 mediated inflammatory pathology. This project has been made possible in part by grant number 2020-216949 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation to Alex K. Shalek and Jose Ordovas-Montanes. Carly G. K. Ziegler was supported by T32GM007753 from the NIH. Jose Ordovas-Montanes is a New York Stem Cell Foundation – Robertson Investigator. Jose Ordovas-Montanes was supported by the Richard and Susan Smith Family Foundation, the AGA Research Foundation’s AGA-Takeda Pharmaceuticals Research Scholar Award in IBD – AGA2020-13-01, the Food Allergy Science Initiative, and The New York Stem Cell Foundation. Bruce H. Horwitz was supported by DK122532-01A1, NIH; 12019PG-CD002, The Leona M. and Harry B. Helmsley Charitable Trust; SRA #54518, and Crohn’s and Colitis Foundation. P.L. was supported by 5T32AG000222 from the NIH. Andrew W. Navia was supported by the Ludwig Center for Molecular Oncology at MIT. Alex K. Shalek was supported by the Bill and Melinda Gates Foundation, Sloan Fellowship in Chemistry, the NIH (5U24AI118672), and the Ragon Institute of MGH, MIT and Harvard.

CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. Tounderstand the molecular basis for Cas13d function and explain its compact molecular architecture, weresolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3Å resolution, respectively. Furthermore, a 6.5Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.

CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function, we resolved cryo-electron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, explain the compact molecular architecture of Cas13d and provide insights into the structural transitions required for enzyme activation. Our comprehensive analysis of Cas13d in diverse enzymatic states facilitated site-specific truncations for minimal size and delineates a blueprint for improving biomolecular applications of RNA targeting.

Summary Class 2 CRISPR-Cas systems endow microbes with diverse mechanisms for adaptive immunity. Here, we analyzed prokaryotic genome and metagenome sequences to identify an uncharacterized family of RNA-guided, RNA-targeting CRISPR systems that we classify as type VI-D. Biochemical characterization and protein engineering of seven distinct orthologs generated a ribonuclease effector derived from Ruminococcus flavefaciens XPD3002 (CasRx) with robust activity in human cells. CasRx-mediated knockdown exhibits high efficiency and specificity relative to RNA interference across diverse endogenous transcripts. As one of the most compact single-effector Cas enzymes, CasRx can also be flexibly packaged into adeno-associated virus. We target virally encoded, catalytically inactive CasRx to cis elements of pre-mRNA to manipulate alternative splicing, alleviating dysregulated tau isoform ratios in a neuronal model of frontotemporal dementia. Our results present CasRx as a programmable RNA-binding module for efficient targeting of cellular RNA, enabling a general platform for transcriptome engineering and future therapeutic development.