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Transthyretin amyloidosis with cardiomyopathy (ATTR-CM) is a progressive, often fatal disease. Nexiguran ziclumeran (nex-z) is an investigational therapy based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease) targeting the gene encoding transthyretin ( ). In this phase 1, open-label trial, we administered a single intravenous infusion of nex-z to patients with ATTR-CM. Primary objectives included assessment of the effect of nex-z on safety and pharmacodynamics, including the serum TTR level. Secondary end points included changes in N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels, high-sensitivity cardiac troponin T levels, the 6-minute walk distance, and the New York Heart Association (NYHA) class. A total of 36 patients received nex-z and completed at least 12 months of follow-up. Of these patients, 50% were in NYHA class III and 31% had variant ATTR-CM. The mean percent change from baseline in the serum TTR level was -89% (95% confidence interval [CI], -92 to -87) at 28 days and -90% (95% CI, -93 to -87) at 12 months. Adverse events were reported in 34 patients. Five had transient infusion-related reactions, and two had transient liver-enzyme elevations that were assessed as treatment-related. Serious adverse events, most of which were consistent with ATTR-CM, were reported in 14 patients. The geometric mean factor change from baseline to month 12 was 1.02 (95% CI, 0.88 to 1.17) in the NT-proBNP level and 0.95 (95% CI, 0.89 to 1.01) in the high-sensitivity cardiac troponin T level. The median change from baseline to month 12 in the 6-minute walk distance was 5 m (interquartile range, -33 to 49). A total of 92% of the patients had either improvement or no change in their NYHA class. In this phase 1 study involving patients with ATTR-CM, treatment with a single dose of nex-z was associated with transient infusion-related reactions and consistent, rapid, and durable reductions in serum TTR levels. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).

Using whole-cell phenotypic assays, the GlaxoSmithKline high-throughput screening (HTS) diversity set of 1.8 million compounds was screened against the three kinetoplastids most relevant to human disease, i.e. Leishmania donovani , Trypanosoma cruzi and Trypanosoma brucei . Secondary confirmatory and orthogonal intracellular anti-parasiticidal assays were conducted and the potential for non-specific cytotoxicity determined. Hit compounds were chemically clustered and triaged for desirable physicochemical properties. The hypothetical biological target space covered by these diversity sets was investigated through bioinformatics methodologies. Consequently, three anti-kinetoplastid chemical boxes of ~200 compounds each were assembled. Functional analyses of these compounds suggest a wide array of potential modes of action against kinetoplastid kinases, proteases and cytochromes as well as potential host–pathogen targets. This is the first published parallel high throughput screening of a pharma compound collection against kinetoplastids. The compound sets are provided as an open resource for future lead discovery programs and to address important research questions.

Kinetoplastids are a group of flagellated protozoans that include the species Trypanosoma and Leishmania, which are human pathogens with devastating health and economic effects. The sequencing of the genomes of some of these species has highlighted their genetic relatedness and underlined differences in the diseases that they cause. As we discuss in this Review, steady progress using a combination of molecular, genetic, immunologic, and clinical approaches has substantially increased understanding of these pathogens and important aspects of the diseases that they cause. Consequently, the paths for developing additional measures to control these \"neglected diseases\" are becoming increasingly clear, and we believe that the opportunities for developing the drugs, diagnostics, vaccines, and other tools necessary to expand the armamentarium to combat these diseases have never been better.

CRISPR gene editing holds great promise to modify DNA sequences in somatic cells to treat disease. However, standard computational and biochemical methods to predict off-target potential focus on reference genomes. We developed an efficient tool called CRISPRme that considers single-nucleotide polymorphism (SNP) and indel genetic variants to nominate and prioritize off-target sites. We tested the software with a BCL11A enhancer targeting guide RNA (gRNA) showing promise in clinical trials for sickle cell disease and β-thalassemia and found that the top candidate off-target is produced by an allele common in African-ancestry populations (MAF 4.5%) that introduces a protospacer adjacent motif (PAM) sequence. We validated that SpCas9 generates strictly allele-specific indels and pericentric inversions in CD34 + hematopoietic stem and progenitor cells (HSPCs), although high-fidelity Cas9 mitigates this off-target. This report illustrates how genetic variants should be considered as modifiers of gene editing outcomes. We expect that variant-aware off-target assessment will become integral to therapeutic genome editing evaluation and provide a powerful approach for comprehensive off-target nomination. CRISPRme is an off-target nomination tool that accounts for human genetic diversity. Ancestry-dependent allele-specific off-target edits can occur with therapies currently in clinical trials, highlighting the importance of genetic variation-aware assessment.

Clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated 9 (Cas9) genome editing is revolutionizing fundamental research and has great potential for the treatment of many diseases. While editing of immortalized cell lines has become relatively easy, editing of therapeutically relevant primary cells and tissues can remain challenging. One recent advancement is the delivery of a Cas9 protein and an in vitro-transcribed (IVT) guide RNA (gRNA) as a precomplexed ribonucleoprotein (RNP). This approach allows editing of primary cells such as T cells and hematopoietic stem cells, but the consequences beyond genome editing of introducing foreign Cas9 RNPs into mammalian cells are not fully understood. Here, we show that the IVT gRNAs commonly used by many laboratories for RNP editing trigger a potent innate immune response that is similar to canonical immune-stimulating ligands. IVT gRNAs are recognized in the cytosol through the retinoic acid-inducible gene I (RIG-I) pathway but not the melanoma differentiation-associated gene 5 (MDA5) pathway, thereby triggering a type I interferon response. Removal of the 5'-triphosphate from gRNAs ameliorates inflammatory signaling and prevents the loss of viability associated with genome editing in hematopoietic stem cells. The potential for Cas9 RNP editing to induce a potent antiviral response indicates that care must be taken when designing therapeutic strategies to edit primary cells.

The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 Å crystal structure of Kluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded and processed by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1–8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2′-hydroxyl groups pre-organizing the backbone of nucleotides 2–8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide–target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage. Argonaute proteins are an essential part of the guide-RNA–protein complex that carries out RNA-induced gene silencing; structure–function studies of the yeast complex reveal conserved features of the eukaryotic complex, which underlie formation of the catalytically active conformation. Chance meeting for Argonaute The functional complex that carries out RNA-induced gene silencing consists of an Argonaute (Ago) protein bound to a short single-stranded guide RNA. This complex recognizes and binds a complementary messenger RNA sequence and mediates either RNA cleavage or repression of its translation. The 3.2-Ångström crystal structure of Kluyveromyces polysporus Argonaute, bound by chance to nonspecific guide RNA, has now been determined. The prokaryotic and human Ago protein structures have been solved in inactive conformations, but this yeast Ago structure is in an active conformation that reveals the mechanism of catalysis.

Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR–Cas9 or CRISPR–Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks. A PAMless CRISPR nuclease is applied for precision DNA cleavage and cloning.

Endosymbiotic relationships between eukaryotic and prokaryotic cells are common in nature. Endosymbioses between two eukaryotes are also known; cyanobacterium-derived plastids have spread horizontally when one eukaryote assimilated another. A unique instance of a non-photosynthetic, eukaryotic endosymbiont involves members of the genus Paramoeba , amoebozoans that infect marine animals such as farmed fish and sea urchins. Paramoeba species harbor endosymbionts belonging to the Kinetoplastea, a diverse group of flagellate protists including some that cause devastating diseases. To elucidate the nature of this eukaryote-eukaryote association, we sequenced the genomes and transcriptomes of Paramoeba pemaquidensis and its endosymbiont Perkinsela sp. The endosymbiont nuclear genome is ~9.5 Mbp in size, the smallest of a kinetoplastid thus far discovered. Genomic analyses show that Perkinsela sp. has lost the ability to make a flagellum but retains hallmark features of kinetoplastid biology, including polycistronic transcription, trans -splicing, and a glycosome-like organelle. Mosaic biochemical pathways suggest extensive ‘cross-talk’ between the two organisms, and electron microscopy shows that the endosymbiont ingests amoeba cytoplasm, a novel form of endosymbiont-host communication. Our data reveal the cell biological and biochemical basis of the obligate relationship between Perkinsela sp. and its amoeba host, and provide a foundation for understanding pathogenicity determinants in economically important Paramoeba .

Background The Euglenozoa are a protist group with an especially rich history of evolutionary diversity. They include diplonemids, representing arguably the most species-rich clade of marine planktonic eukaryotes; trypanosomatids, which are notorious parasites of medical and veterinary importance; and free-living euglenids. These different lifestyles, and particularly the transition from free-living to parasitic, likely require different metabolic capabilities. We carried out a comparative genomic analysis across euglenozoan diversity to see how changing repertoires of enzymes and structural features correspond to major changes in lifestyles. Results We find a gradual loss of genes encoding enzymes in the evolution of kinetoplastids, rather than a sudden decrease in metabolic capabilities corresponding to the origin of parasitism, while diplonemids and euglenids maintain more metabolic versatility. Distinctive characteristics of molecular machines such as kinetochores and the pre-replication complex that were previously considered specific to parasitic kinetoplastids were also identified in their free-living relatives. Therefore, we argue that they represent an ancestral rather than a derived state, as thought until the present. We also found evidence of ancient redundancy in systems such as NADPH-dependent thiol-redox. Only the genus Euglena possesses the combination of trypanothione-, glutathione-, and thioredoxin-based systems supposedly present in the euglenozoan common ancestor, while other representatives of the phylum have lost one or two of these systems. Lastly, we identified convergent losses of specific metabolic capabilities between free-living kinetoplastids and ciliates. Although this observation requires further examination, it suggests that certain eukaryotic lineages are predisposed to such convergent losses of key enzymes or whole pathways. Conclusions The loss of metabolic capabilities might not be associated with the switch to parasitic lifestyle in kinetoplastids, and the presence of a highly divergent (or unconventional) kinetochore machinery might not be restricted to this protist group. The data derived from the transcriptomes of free-living early branching prokinetoplastids suggests that the pre-replication complex of Trypanosomatidae is a highly divergent version of the conventional machinery. Our findings shed light on trends in the evolution of metabolism in protists in general and open multiple avenues for future research.

The genus Leishmania includes approximately 53 species, 20 of which cause human leishmaniais; a significant albeit neglected tropical disease. Leishmaniasis has afflicted humans for millennia, but how ancient is Leishmania and where did it arise? These questions have been hotly debated for decades and several theories have been proposed. One theory suggests Leishmania originated in the Palearctic, and dispersed to the New World via the Bering land bridge. Others propose that Leishmania evolved in the Neotropics. The Multiple Origins theory suggests that separation of certain Old World and New World species occurred due to the opening of the Atlantic Ocean. Some suggest that the ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia evolved on Gondwana between 90 and 140 million years ago. In the present study a detailed molecular and morphological characterisation was performed on a novel Australian trypanosomatid following its isolation in Australia's tropics from the native black fly, Simulium (Morops) dycei Colbo, 1976. Phylogenetic analyses were conducted and confirmed this parasite as a sibling to Zelonia costaricensis, a close relative of Leishmania previously isolated from a reduviid bug in Costa Rica. Consequently, this parasite was assigned the name Zelonia australiensis sp. nov. Assuming Z. costaricensis and Z. australiensis diverged when Australia and South America became completely separated, their divergence occurred between 36 and 41 million years ago at least. Using this vicariance event as a calibration point for a phylogenetic time tree, the common ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia appeared in Gondwana approximately 91 million years ago. Ultimately, this study contributes to our understanding of trypanosomatid diversity, and of Leishmania origins by providing support for a Gondwanan origin of dixenous parasitism in the Leishmaniinae.