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Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes. Cas12a is widely used in diagnostic platforms. Here the authors show that Cas12a can be programmed to directly detect RNA substrates, this is due to the 3’-end of the crRNA tolerating both RNA and DNA substrates: they use this to report a method, SAHARA, to detect RNA sequences.

Antibiotic resistance genes (ARGs) are widespread among bacteria. However, not all ARGs pose serious threats to public health, highlighting the importance of identifying those that are high-risk. Here, we developed an ‘omics-based’ framework to evaluate ARG risk considering human-associated-enrichment, gene mobility, and host pathogenicity. Our framework classifies human-associated, mobile ARGs (3.6% of all ARGs) as the highest risk, which we further differentiate as ‘current threats’ (Rank I; 3%) - already present among pathogens - and ‘future threats’ (Rank II; 0.6%) - novel resistance emerging from non-pathogens. Our framework identified 73 ‘current threat’ ARG families. Of these, 35 were among the 37 high-risk ARGs proposed by the World Health Organization and other literature; the remaining 38 were significantly enriched in hospital plasmids. By evaluating all pathogen genomes released since framework construction, we confirmed that ARGs that recently transferred into pathogens were significantly enriched in Rank II (‘future threats’). Lastly, we applied the framework to gut microbiome genomes from fecal microbiota transplantation donors. We found that although ARGs were widespread (73% of genomes), only 8.9% of genomes contained high-risk ARGs. Our framework provides an easy-to-implement approach to identify current and future antimicrobial resistance threats, with potential clinical applications including reducing risk of microbiome-based interventions. Antibiotic resistance genes are common but not all are of high risk to human health. Here, the authors develop an omics-based framework for ranking genes by risk that incorporates level of enrichment in human associated environments, gene mobility, and host pathogenicity.

Cognitive deficits are a core feature of schizophrenia, account for much of the impaired functioning associated with the disorder and are not responsive to existing treatments. In this review, we first describe the clinical presentation and natural history of these deficits. We then consider aetiological factors, highlighting how a range of similar genetic and environmental factors are associated with both cognitive function and schizophrenia. We then review the pathophysiological mechanisms thought to underlie cognitive symptoms, including the role of dopamine, cholinergic signalling and the balance between GABAergic interneurons and glutamatergic pyramidal cells. Finally, we review the clinical management of cognitive impairments and candidate novel treatments.

Most genetic variants that contribute to disease 1 are challenging to correct efficiently and without excess byproducts 2 , 3 , 4 – 5 . Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB ) and Tay–Sachs disease (requiring a deletion in HEXA ); to install a protective transversion in PRNP ; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases. A new DNA-editing technique called prime editing offers improved versatility and efficiency with reduced byproducts compared with existing techniques, and shows potential for correcting disease-associated mutations.

Exosomes and other small extracellular vesicles (sEVs) provide a unique mode of cell-to-cell communication in which microRNAs (miRNAs) produced and released from one cell are taken up by cells at a distance where they can enact changes in gene expression 1 – 3 . However, the mechanism by which miRNAs are sorted into exosomes/sEVs or retained in cells remains largely unknown. Here we demonstrate that miRNAs possess sorting sequences that determine their secretion in sEVs (EXOmotifs) or cellular retention (CELLmotifs) and that different cell types, including white and brown adipocytes, endothelium, liver and muscle, make preferential use of specific sorting sequences, thus defining the sEV miRNA profile of that cell type. Insertion or deletion of these CELLmotifs or EXOmotifs in a miRNA increases or decreases retention in the cell of production or secretion into exosomes/sEVs. Two RNA-binding proteins, Alyref and Fus, are involved in the export of miRNAs carrying one of the strongest EXOmotifs, CGGGAG. Increased miRNA delivery mediated by EXOmotifs leads to enhanced inhibition of target genes in distant cells. Thus, this miRNA code not only provides important insights that link circulating exosomal miRNAs to tissues of origin, but also provides an approach for improved targeting in RNA-mediated therapies. MicroRNAs encode sorting sequences that determine whether they are secreted in exosomal vesicles to regulate gene expression in distant cells or retained in cells that produced them, with different sequences used by individual cell types.

Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies 1 – 7 . However, CAR-T cell therapy currently has several limitations 8 – 12 . Here we successfully developed a two-in-one approach to generate non-viral, gene-specific targeted CAR-T cells through CRISPR–Cas9. Using the optimized protocol, we demonstrated feasibility in a preclinical study by inserting an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and showed superior ability to eradicate tumour cells in xenograft models. In adoptive therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma (ClinicalTrials.gov, NCT04213469 ), we observed a high rate (87.5%) of complete remission and durable responses without serious adverse events in eight patients. Notably, these enhanced CAR-T cells were effective even at a low infusion dose and with a low percentage of CAR + cells. Single-cell analysis showed that the electroporation method resulted in a high percentage of memory T cells in infusion products, and PD1 interference enhanced anti-tumour immune functions, further validating the advantages of non-viral, PD1 -integrated CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, gene-specific integrated CAR-T cells, thus providing an innovative technology for CAR-T cell therapy. Non-viral CAR-T cells with gene-specific targeted integration are safe and effective in patients with lymphoma.

Trimethylation of histone H3 lysine 4 (H3K4me3) is associated with transcriptional start sites and has been proposed to regulate transcription initiation 1 , 2 . However, redundant functions of the H3K4 SET1/COMPASS methyltransferase complexes complicate the elucidation of the specific role of H3K4me3 in transcriptional regulation 3 , 4 . Here, using mouse embryonic stem cells as a model system, we show that acute ablation of shared subunits of the SET1/COMPASS complexes leads to a complete loss of all H3K4 methylation. Turnover of H3K4me3 occurs more rapidly than that of H3K4me1 and H3K4me2 and is dependent on KDM5 demethylases. Notably, acute loss of H3K4me3 does not have detectable effects on transcriptional initiation but leads to a widespread decrease in transcriptional output, an increase in RNA polymerase II (RNAPII) pausing and slower elongation. We show that H3K4me3 is required for the recruitment of the integrator complex subunit 11 (INTS11), which is essential for the eviction of paused RNAPII and transcriptional elongation. Thus, our study demonstrates a distinct role for H3K4me3 in transcriptional pause-release and elongation rather than transcriptional initiation. Acute loss of H3K4me3 does not have detectable effects on transcriptional initiation, but leads to a widespread decrease in transcriptional output, an increase in RNA polymerase II pausing and slower elongation

Epigenetic research has accelerated rapidly in the twenty-first century, generating justified excitement and hope, but also a degree of hype. Here we review how the field has evolved over the last few decades and reflect on some of the recent advances that are changing our understanding of biology. We discuss the interplay between epigenetics and DNA sequence variation as well as the implications of epigenetics for cellular memory and plasticity. We consider the effects of the environment and both intergenerational and transgenerational epigenetic inheritance on biology, disease and evolution. Finally, we present some new frontiers in epigenetics with implications for human health. The authors review recent advances and current debates in epigenetics, including how epigenetic mechanisms interact with genetic variation, ageing, disease and the environment.

The unique capabilities of CRISPR technologies have enabled a broad range of applications in biomedicine and agriculture. Programmable DNA cleavage using CRISPR–Cas9 enables efficient, site-specific genome engineering in single cells and whole organisms. In the research arena, versatile CRISPR-enabled genome editing has been used in various ways, such as controlling transcription, modifying epigenomes, conducting genome-wide screens and imaging chromosomes. CRISPR systems are already being used to alleviate genetic disorders in animals and are likely to be employed soon in the clinic to treat human diseases of the eye and blood. Two clinical trials using CRISPR-Cas9 for targeted cancer therapies have been approved in China and the United States. Beyond biomedical applications, these tools are now being used to expedite crop and livestock breeding, engineer new antimicrobials and control disease-carrying insects with gene drives.

Self-organizing neural organoids grown from pluripotent stem cells 1 – 3 combined with single-cell genomic technologies provide opportunities to examine gene regulatory networks underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin data over a dense time course in human organoids covering neuroepithelial formation, patterning, brain regionalization and neurogenesis, and identify temporally dynamic and brain-region-specific regulatory regions. We developed Pando—a flexible framework that incorporates multi-omic data and predictions of transcription-factor-binding sites to infer a global gene regulatory network describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoids. We find that certain factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. We show that the transcription factor GLI3 is required for cortical fate establishment in humans, recapitulating previous research performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify two distinct GLI3 regulomes that are central to telencephalic fate decisions: one regulating dorsoventral patterning with HES4/5 as direct GLI3 targets, and one controlling ganglionic eminence diversification later in development. Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology. A multi-omic atlas of brain organoid development facilitates the inference of an underlying gene regulatory network using the newly developed Pando framework and shows—in conjunction with perturbation experiments—that GLI3 controls forebrain fate establishment through interaction with HES4/5 regulomes.