Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

Background: Weeks after SARS-CoV-2 infection or exposure, some children develop a severe, life-threatening illness called Multisystem Inflammatory Syndrome in Children (MIS-C).Gastrointestinal symptoms are common in MIS-C patients and severe hyperinflammatory response ensues with potential for cardiac complications. The cause of MIS-C has not previously been identified.Methods: Here, we analyzed biospecimens from 100 children: 19 children with MIS-C, 26 with acute COVID-19, and 55 controls. Stool was assessed for SARS-CoV-2 by RT-PCR and plasma was assessed for markers of breakdown of mucosal barrier integrity, including zonulin.Ultrasensitive antigen detection was used to probe for SARS-CoV-2 antigenemia in plasma, and immune responses were characterized. As proof of concept, we treated a MIS-C patient with larazotide, a zonulin antagonist, and monitored impact on antigenemia and clinical response. Results:We showed that in MIS-C, prolonged presence of SARS-CoV-2 in the GI tract leads to release of zonulin, a biomarker of intestinal permeability, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation. The MIS-C patient treated with larazotide displayed a coinciding decrease in plasma SARS-CoV-2 Spike antigen levels, inflammatory markers, and a resultant clinical improvement above that achieved with currently available treatments. Conclusion:These mechanistic data of MIS-C pathogenesis provide insight into targets for diagnosing, treating, and preventing MIS-C, which are urgently needed for this increasingly common severe COVID-19-related disease in children.

Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.

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