CRISPR-associated (Cas) transposases (CAST) are RNA-guided systems capable of programmable integration of large segments of DNA without creating double-strand breaks. Engineered Cascade CAST function in human cells but are challenging to deploy due to the complexity of the targeting components. Unlike Cascade, which require three Cas proteins, type V-K CAST require a single Cas12k effector for targeting. Here, we show that compact type V-K CAST from uncultivated microbes are repurposable for programmable DNA integration into the genome of human cells. Engineering for nuclear localization and function enables integration of a therapeutically relevant transgene at a safe-harbor site in multiple human cell types. Notably, off-targets are rare events reproducibly found in specific genomic regions. These CAST advancements are expected to accelerate applications of genome editing to therapeutic development, biotechnology, and synthetic biology.

The discovery of immune checkpoints and the rapid growth of immuno-oncology have sparked efforts to utilize the immune system to treat a wide range of cancer types/subtypes. Although the major focus of immuno-oncology over the past decades has been to manipulate the adaptive immune system, recent attention has been given to manipulating the innate immune system to treat cancer and/or to enhance adaptive responses. In this study, we detailed the intracellular protein dual specificity phosphatase 11 (DUSP11) as an innate immune checkpoint in non-small cell lung cancer adenocarcinoma (LUAD). The expression of this atypical phosphatase was correlated with patient survival for multiple cancer types, and we reported here that its activity was important for the viability of lung cancer cells in vitro. Specifically, we demonstrated that DUSP11 knockdown in LUAD cells induces apoptosis and an innate immune response capable of activating other cells in vitro, and we provided evidence that these phenotypes are primarily mediated by the pattern recognition receptor, retinoic acid-inducible gene I. Finally, we showed that the expression of DUSP11 was important for tumor engraftment and growth of human LUAD in mice. Overall, these data are the first to establish DUSP11 as an immunosuppressive, pro-neoplastic, and potentially targetable protein in LUAD. In addition, our data suggest that the anticancer mechanisms induced by diminishing the activity of DUSP11 are likely to be generalizable to other cancer types such as breast and skin cancers, warranting future investigation and highlighting therapeutic potential.

Protein aggregation is linked to many chronic and devastating neurodegenerative human diseases and is strongly associated with aging. This work demonstrates that protein aggregation and oligomerization can be evaluated by a solid-state nanopore method at the single molecule level. A silicon nitride nanopore sensor was used to characterize both the amyloidogenic and native-state oligomerization of a model protein ß-lactoglobulin variant A (βLGa). The findings from the nanopore measurements are validated against atomic force microscopy (AFM) and dynamic light scattering (DLS) data, comparing βLGa aggregation from the same samples at various stages. By calibrating with linear and circular dsDNA, this study estimates the amyloid fibrils’ length and diameter, the quantity of the βLGa aggregates, and their distribution. The nanopore results align with the DLS and AFM data and offer additional insight at the level of individual protein molecular assemblies. As a further demonstration of the nanopore technique, βLGa self-association and aggregation at pH 4.6 as a function of temperature were measured at high (2 M KCl) and low (0.1 M KCl) ionic strength. This research highlights the advantages and limitations of using solid-state nanopore methods for analyzing protein aggregation.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

Non-small cell lung cancer (NSCLC) adenocarcinoma (LUAD) is a leading cause of death worldwide. Activating mutations in the tyrosine kinase domain of the oncogene epidermal growth factor receptor (EGFR) are responsible for similar to 10-50% of all LUAD cases. Although tyrosine kinase inhibitors (TKIs) have been effective in prolonging patient survival and quality of life, acquired resistance and disease progression are inevitable, presenting a clear unmet need for alternative or adjuvant therapeutics. Here we show that an anti-EGFR aptamer (EGFRapt) decreases viability and tumor growth of LUAD cell lines harboring the L858R +/- T790M mutation in EGFR. Additionally, we elucidate the mechanism by which EGFRapt exerts these effects by monitoring cellular processes associated with kinase-dependent and kinase-independent mechanisms. Overall, these data establish that EGFRapt has direct anti-cancer activity in mutant EGFR positive LUAD via targetable mechanisms that are independent of existing approaches, and they provide a foundation for further development of nucleic acid-based therapies that target EGFR.

A significant fraction of non-small cell lung cancer (NSCLC) cases are due to oncogenic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). Anti-EGFR antibodies have shown limited clinical benefit for NSCLC, whereas tyrosine kinase inhibitors (TKIs) are effective, but resistance ultimately occurs. The current land-scape suggests that alternative ligands that target wild-type and mutant EGFRs are desirable for targeted therapy or drug delivery development. Here we evaluate NSCLC targeting using an anti-EGFR aptamer (MinE07). We demonstrate that interaction sites of MinE07 overlap with clinically relevant antibodies targeting extracellular domain III and that MinE07 retains binding to EGFR harboring the most com-mon oncogenic and resistance mutations. When MinE07 was linked to an anti-c-Met aptamer, the EGFR/c-Met bispe-cific aptamer (bsApt) showed superior labeling of NSCLC cells in vitro relative to monospecific aptamers. However, dual targeting in vivo did not improve the recognition of NSCLC xenografts compared to MinE07. Interestingly, bio-distribution of Cy7-labeled bsApt differed significantly from Alexa Fluor 750-labeled bsApt. Overall, our findings demonstrate that aptamer formulations containing MinE07 can target ectopic lung cancer without additional stabilization or PEGylation and highlights the potential of MinE07 as a targeting reagent for the recognition of NSCLC harboring clinically relevant EGFRs.

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans ( C. elegans ) null for a cysteine protease inhibitor, srp-6 , undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3 , respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans , this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors.

Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of β2-microglobulin, TIGIT, FAS, and PDCD1. Simultaneous double knockout of TRAC and TRBC was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into TRAC (up to ∼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell–based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.

Evasion of immune destruction is a major hallmark of cancer. Recent US Food and Drug Administration (FDA) approvals of various immunomodulating therapies underline the important role that reprogramming the immune system can play in combating this disease. However, a wide range of side effects still limit the therapeutic potential of immunomodulators, suggesting a need for more precise reagents with negligible off-target and on-target/off-tumor effects. Aptamers are single-chained oligonucleotides that bind their targets with high specificity and affinity owing to their three-dimensional (3D) structures, and they are one potential way to address this need. In particular, bispecific aptamers (bsApts) have been shown to induce artificial immune synapses that promote T cell activation and subsequent tumor cell lysis in various in vitro and in vivo pre-clinical models. We discuss these advances here, along with gaps in bsApt biology at both the cellular and resident tissue levels that should be addressed to accelerate their translation into the clinic. The broad application, minimal production cost, and relative lack of immunogenicity of bsApts give them some ideal qualities for manipulating the immune system. Building upon lessons from other novel therapies, bsApts could soon provide clinicians with an immunomodulating toolbox that is not only potent and efficacious but exercises a wide therapeutic index.

Analysis of long timespan heliophysics and space physics data or application of machine learning algorithms can require access to petabyte-scale and larger data sets and sufficient computational capacity to process such "big data ". We provide a summary of Python support and performance statistics for the major scientific data formats under consideration for access to heliophysics data in cloud computing environments. The Heliophysics Data Portal lists 21 different formats used in heliophysics and space physics; our study focuses on Python support for the most-used formats of CDF, FITS, and NetCDF4/HDF. In terms of package support, there is no single Python package that supports all of the common heliophysics file types, while NetCDF/HDF5 is the most supported file type. In terms of technical implementation within a cloud environment, we profile file performance in Amazon Web Services (AWS). Effective use of AWS cloud-based storage requires Python libraries designed to read their S3 storage format. In Python, S3-aware libraries exist for CDF, FITS, and NetCDF4/HDF. The existing libraries use different approaches to handling cloud-based data, each with tradeoffs. With these caveats, Python pairs well with AWS's cloud storage within the current Python ecosystem for existing heliophysics data, and cloud performance in Python is continually improving. We recommend anyone considering cloud use or optimization of data formats for cloud use specifically profile their given data set, as instrument-specific data characteristics have a strong effect on which approach is best for cloud use.

Programmable, RNA-guided nucleases are diverse enzymes that have been repurposed for biotechnological applications. However, to further expand the therapeutic application of these tools there is a need for targetable systems that are small enough to be delivered efficiently. Here, we mined an extensive genome-resolved metagenomics database and identified families of uncharacterized RNA-guided, compact nucleases (between 450 and 1,050 aa). We report that Cas9d, a new CRISPR type II subtype, contains Zinc-finger motifs and high arginine content, features that we also found in nucleases related to HEARO effectors. These enzymes exhibit diverse biochemical characteristics and are broadly targetable. We show that natural Cas9d enzymes are capable of genome editing in mammalian cells with >90% efficiency, and further engineered nickase variants into the smallest base editors active in E. coli and human cells. Their small size, broad targeting potential, and translatability suggest that Cas9d and HEARO systems will enable a variety of genome editing applications.