Biology subjects

Myhrvold, C.

Publications and source records attributed to Myhrvold, C..

18 recordsLinked to original sources

bioRxiv2026

Protein Deimmunization with a Generative Diffusion Model

Protein immunogenicity is a major cause of biologic drug failure, leading to the production of anti-drug antibodies (ADA) that accelerate drug clearance, rejection of repeated doses, abolished efficacy, and sometimes life threatening anaphylaxis in patients. These responses constrain the clinical use of synthetic protein therapeutics from non-human origin. To efficiently deimmunize protein therapeutics, we developed a generative discrete diffusion model (MESSI), which takes protein structures as input and outputs sequences that preserve structure without containing peptides predicted to bind major histocompatibility complex class II molecules. MESSI designs sequences with high structural recovery and down to 0 predicted epitopes for specified MHC molecules at higher efficiency than post-hoc filtering approaches. Experimentally, we first applied MESSI to deimmunize a luciferase and a de novo designed mini-binder against PD-1, resulting in variants with no predicted epitopes and higher activity than their parents. Lastly, we coupled MESSI with evolution guidance to deimmunize uricase, a clinically approved enzyme with severe immunogenicity, and found deimmunized variants with higher catalytic activity than their natural orthologs. MESSI enables generative design of functional proteins with minimized immunogenicity, potentially making protein therapeutics safer and more effective.

bioengineering↗

bioRxiv2026

Intersegmental transfers drive target search in an RNA-targeting CRISPR system

Sequence-specific RNA-binding proteins (RBPs) must efficiently locate their targets among a multitude of cellular RNAs. Cas13, an RNA-guided CRISPR protein, represents an ideal model system in which to study this search process. Cas13 combats bacteriophage infection by cleaving RNA nonspecifically upon binding of its crRNA to the target RNA sequence; thus, Cas13s search for its RNA target comes with a time constraint determined by the rate of phage multiplication. The mechanism by which Cas13 locates its target within this critical window remains unknown. Here, we investigate Cas13s mechanism of target search through integration of biophysical modeling, activity assays, and biochemical characterization. We show that Cas13 employs facilitated diffusion to accelerate its search, and find that Cas13s search time when targeting RNAs of different lengths cannot be explained by 1D sliding, the search mechanism used by many DNA-binding proteins. We propose that Cas13 primarily searches for its RNA target by intersegmental transfers (ITs), non-specifically binding the RNA at two locations and directly switching between them without fully dissociating from the RNA. We develop a biophysical model for ITs in an RNA context that we subsequently validate experimentally. Furthermore, we demonstrate that ITs can differentially accelerate the search process for a broad class of RNA-binding proteins, as opposed to their DNA-binding counterparts, due to RNAs short persistence length and the heterogeneity of RNA lengths in the cell. Our results illuminate how Cas13 achieves rapid target recognition in a complex RNA environment, and implicate ITs as a potentially widespread solution to the RNA search problem.

biophysics↗

bioRxiv2025

A handheld microfluidic manifold for massively multiplexed CRISPR-based nucleic acid detection

Multiplexed methods for nucleic acid detection are immensely challenging to deploy outside of laboratory settings. Conversely, field-deployable methods are limited to low levels of multiplexing. Here, we introduce Scalable On-site Nucleic Acid Testing Architecture (SONATA), enabling >100-chamber reaction partitioning for multiplexed nucleic acid amplification and detection with a portable microfluidic manifold. The manifold directs a diluted sample into individual reaction chambers, each of which contains lyophilized reagents and a small stir bar or bead for mixing. Samples can be loaded using a syringe by hand, greatly simplifying the testing process. We demonstrate the integration of the platform with Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a sensitive and deployable CRISPR-based detection technology. We show that deployed with SONATA, SHINE retains its sensitivity, enabling highly multiplexed pathogen detection in [≤] 1 hour. In addition, we demonstrate the detection of single-nucleotide variants, including mutations associated with drug susceptibility.

bioengineering↗

bioRxiv2025

Temperature-dependent modulation of aberrant influenza A virus RNA synthesis affects innate immune signaling

Fever during influenza A virus (IAV) infection is triggered by the innate immune response. Various factors contribute to this response, including IAV mini viral RNAs (mvRNA), which trigger RIG-I signaling when their replication and transcription are dysregulated by template loops (t-loop). It is presently not well understood whether the fever response to IAV infection impacts subsequent viral replication and innate immune activation. Here we show that IAV infection at temperatures that simulate fever leads to increased antiviral signaling in H1N1 and H3N2 infections. Mathematical modeling and experimental analyses reveal that differential IAV nucleoprotein and RNA polymerase production increase mvRNA and interferon production. Moreover, at the higher infection temperature, mvRNAs with dysregulating t-loops contribute most to the innate immune activation. We propose that fever during IAV infection can establish a positive feedback loop in which elevated aberrant RNA synthesis and innate immune activation can contribute to the dysregulation of cytokine production.

microbiology↗

bioRxiv2025

Amplification-free Detection of Zoonotic Viruses Using Cas13 and Multiple CRISPR RNAs

Zoonotic viruses such as hantaviruses and influenza A viruses present a threat to humans and livestock. There is thus a need for methods that are rapid, sensitive, and relatively cheap to detect infections with these pathogens early. Here we use an amplification-free CRISPR-Cas13-based assay, which is simple, cheap and field-deployable, to detect the presence or absence of genomic hantavirus or influenza A virus RNA. In addition, we evaluate whether the use of multiple CRISPR RNAs (crRNAs) can improve the sensitivity of this amplification-free method. We demonstrate that for the hantaviruses Tula Virus (TULV) and Andes Virus (ANDV) a combination of two or three crRNAs provides the best sensitivity for detecting viral RNA, whereas for influenza virus RNA detection, additional crRNAs provide no benefit. We also show that the amplification-free method can be used to detect TULV and ANDV RNA in tissue culture infection samples and influenza A virus RNA in clinical nasopharyngeal swabs. In clinical samples, the Cas13 assay has an 85% agreement with RT-qPCR for identifying a positive sample. Overall, these findings indicate that amplification-free CRISPR-Cas13 detection of viral RNA has potential as a tool for rapidly detecting zoonotic virus infections.

microbiology↗

bioRxiv2024

Leveraging CRISPR-Cas13d in an inducible knockdown system to interrogate Drosophila germ granule mRNAs

Ribonucleoprotein (RNP) germ granules are hallmarks of germ cells across the animal kingdom and are thought to be hubs for post-transcriptional regulation that promote formation of the germ cell precursors. While numerous RNAs are associated with germ granules in Drosophila, the functions of many in germline development are poorly understood. Current methods for RNA knockdown, such as RNAi, do not allow local depletion of transcripts such as those found in the germ granules. We leveraged CRISPR-Cas13 to create a subcellular RNA knockdown system and tested it on two mRNAs, nanos (nanos) and sarah (sra), whose abundance in germ granules differs. Because Cas13 has both cis and trans cleavage activities, we evaluated the effect of target abundance on off-target RNA depletion. We show on and off-target RNA depletion is coupled when targeting the more abundant nanos germ granule transcripts. Off-target RNA knockdown is less potent when the system is used for less abundant sra transcripts. When sra is knocked down in germ granules, we observed an increase in the calcium indicator GCaMP at the posterior and defective primordial germ cell migration, consistent with sra encoding a negative regulator of calcium signaling. In sum, we report an in vivo Cas13-based system for subcellular knockdown, evaluate its feasibility, and uncover a novel function for sra germ granule transcripts in promoting germline development.

molecular biology↗

bioRxiv2024

Efficient Genome Editing with Chimeric Oligonucleotide-Directed Editing

Prime editing has emerged as a precise and powerful genome editing tool, offering a favorable gene editing profile compared to other Cas9-based approaches. Here we report new nCas9-DNA polymerase fusion proteins to create chimeric oligonucleotide-directed editing (CODE) systems for search-and-replace genome editing. Through successive rounds of engineering, we developed CODEMax and CODEMax(exo+) editors that achieve efficient genome modifications in human cells with low unintended edits. CODEMax and CODEMax(exo+) contain an engineered Bst DNA polymerase derivative known for its robust strand displacement ability. Additionally, CODEMax(exo+) features a 5 to 3 exonuclease activity that promotes effective strand invasion and repair outcomes favoring the incorporation of the desired edit. We demonstrate CODEs can perform small insertions, deletions, and substitutions with improved efficiency compared to PEMax at many loci. Overall, CODEs complement existing prime editors to expand the toolbox for genome manipulations without double-stranded breaks.

bioengineering↗

bioRxiv2023

P. aeruginosa controls both C. elegans attraction and pathogenesis by regulating nitrogen assimilation

Detecting chemical signals is important for identifying food sources and avoiding harmful agents. Like most animals, C. elegans use olfaction to chemotax towards their main food source, bacteria. However, little is known about the bacterial compounds governing C. elegans attraction to bacteria and the physiological importance of these compounds to bacteria. Here, we address these questions by investigating the function of a small RNA, P11, in the pathogen, Pseudomonas aeruginosa, that was previously shown to mediate learned pathogen avoidance. We discovered that this RNA also affects the attraction of untrained C. elegans to P. aeruginosa and does so by controlling production of ammonia, a volatile odorant produced during nitrogen assimilation. We untangle the complex regulation of P. aeruginosa nitrogen assimilation, which is mediated by a partner-switching mechanism involving environmental nitrates, sensor proteins, and P11. In addition to mediating C. elegans attraction, nitrogen assimilation is important for bacterial fitness and pathogenesis during C. elegans infection by P. aeruginosa. These studies define ammonia as a major mediator of trans-kingdom signaling, reveal the physiological importance of nitrogen assimilation for both bacteria and host organisms, and highlight how a bacterial metabolic pathway can either benefit or harm a host in different contexts.

microbiology↗

bioRxiv2023

Cas13-based amplification-free quantification of aberrant RNAs during influenza virus infection

Influenza A virus RNA synthesis produces full-length and aberrant RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that aberrant RNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Understanding the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for understanding their function in IAV infection. Here, we use an amplification-free LbuCas13a-based detection method to quantify mvRNA amplification dynamics and subcellular distributions. We show that our assay can quantify the copy numbers of specific mvRNA sequences in infected tissue culture cells, ferret upper and lower respiratory tract tissue infected with two different pandemic H1N1 IAV strains, or clinical nasopharyngeal swab extracts of hospitalized patients infected with seasonal H1N1 or H3N2 strains. In addition, we find dynamic differences between immunostimulatory and non-immunostimulatory mvRNAs, as well as among mvRNAs derived from different segments, during IAV infection. Overall, our results reveal a hitherto hidden diversity in the behavior of IAV mvRNAs and suggest that individual aberrant RNAs are not produced stochastically.

microbiology↗

bioRxiv2023

RNA structure modulates Cas13 activity and enables mismatch detection

The RNA-targeting CRISPR nuclease Cas13 has emerged as a powerful tool for applications ranging from nucleic acid detection to transcriptome engineering and RNA imaging1-6. Cas13 is activated by the hybridization of a CRISPR RNA (crRNA) to a complementary single-stranded RNA (ssRNA) protospacer in a target RNA1,7. Though Cas13 is not activated by double-stranded RNA (dsRNA) in vitro, it paradoxically demonstrates robust RNA targeting in environments where the vast majority of RNAs are highly structured2,8. Understanding Cas13s mechanism of binding and activation will be key to improving its ability to detect and perturb RNA; however, the mechanism by which Cas13 binds structured RNAs remains unknown9. Here, we systematically probe the mechanism of LwaCas13a activation in response to RNA structure perturbations using a massively multiplexed screen. We find that there are two distinct sequence-independent modes by which secondary structure affects Cas13 activity: structure in the protospacer region competes with the crRNA and can be disrupted via a strand-displacement mechanism, while structure in the region 3 to the protospacer has an allosteric inhibitory effect. We leverage the kinetic nature of the strand displacement process to improve Cas13-based RNA detection, enhancing mismatch discrimination by up to 50-fold and enabling sequence-agnostic mutation identification at low (<1%) allele frequencies. Our work sets a new standard for CRISPR-based nucleic acid detection and will enable intelligent and secondary-structure-guided target selection while also expanding the range of RNAs available for targeting with Cas13.

molecular biology↗

bioRxiv2023

Model-directed generation of CRISPR-Cas13a guide RNAs designs artificial sequences that improve nucleic acid detection

Generating maximally-fit biological sequences has the potential to transform CRISPR guide RNA design as it has other areas of biomedicine. Here, we introduce model-directed exploration algorithms (MEAs) for designing maximally-fit, artificial CRISPR-Cas13a guides--with multiple mismatches to any natural sequence--that are tailored for desired properties around nucleic acid diagnostics. We find that MEA-designed guides offer more sensitive detection of diverse pathogens and discrimination of pathogen variants compared to guides derived directly from natural sequences, and illuminate interpretable design principles that broaden Cas13a targeting.

genomics↗

bioRxiv2023

Highly multiplexed mRNA quantitation with CRISPR-Cas13

RNA quantitation tools are often either high-throughput or cost-effective, but rarely are they both. Existing methods can profile the transcriptome at great expense or are limited to quantifying a handful of genes by labor constraints. A technique that permits more throughput at a reduced cost could enable multi-gene kinetic studies, gene regulatory network analysis, and combinatorial genetic screens. Here, we introduce quantitative Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (qCARMEN): an RNA quantitation technique which leverages the programmable RNA-targeting capabilities of CRISPR-Cas13 to address this challenge by quantifying over 4,500 gene-sample pairs in a single experiment. Using qCARMEN, we studied the response profiles of interferon-stimulated genes (ISGs) during interferon (IFN) stimulation and flavivirus infection. Additionally, we observed isoform switching kinetics during epithelial-mesenchymal transition. qCARMEN is a simple and inexpensive technique that greatly enhances the scalability of RNA quantitation for novel applications with performance similar to gold-standard methods.

molecular biology↗

bioRxiv2023

A small RNA from a natural bacterial pathogen of C. elegans induces transgenerational inheritance of learned avoidance

Previously, we discovered that a small RNA from a clinical isolate of Pseudomonas aeruginosa, PA14, induces learned avoidance and its transgenerational inheritance in C. elegans. Pseudomonas aeruginosa is an important human pathogen, and there are other Pseudomonads in C. elegans natural habitat, but it is unclear whether C. elegans ever encounters PA14-like bacteria in the wild. Thus, it is not known if small RNAs from bacteria found in C. elegans natural habitat can also regulate host behavior and produce heritable behavioral effects. Here we found that a pathogenic Pseudomonas vranovensis strain isolated from the C. elegans microbiota, GRb0427, like PA14, regulates worm behavior: worms learn to avoid this pathogenic bacterium following exposure to GRb0427, and this learned avoidance is inherited for four generations. The learned response is entirely mediated by bacterially-produced small RNAs, which induce avoidance and transgenerational inheritance, providing further support that such mechanisms of learning and inheritance exist in the wild. Using bacterial small RNA sequencing, we identified Pv1, a small RNA from GRb0427, that matches the sequence of C. elegans maco-1. We find that Pv1 is both necessary and sufficient to induce learned avoidance of Grb0427. However, Pv1 also results in avoidance of a beneficial microbiome strain, P. mendocina; this potentially maladaptive response may favor reversal of the transgenerational memory after a few generations. Our findings suggest that bacterial small RNA-mediated regulation of host behavior and its transgenerational inheritance are functional in C. elegans natural environment, and that different bacterial small RNA-mediated regulation systems evolved independently but define shared molecular features of bacterial small RNAs that produce transgenerationally-inherited effects.

genetics↗

bioRxiv2021

Multiplexed detection of bacterial nucleic acids using Cas13 in droplet microarrays

Rapid and accurate diagnosis of infections is fundamental to individual patient care and public health management. Nucleic acid detection methods are critical to this effort, but are limited either in the breadth of pathogens targeted or by the expertise and infrastructure required. We present here a high-throughput system that enables rapid identification of bacterial pathogens, bCARMEN, which utilizes: (1) modular CRISPR-Cas13-based nucleic acid detection with enhanced sensitivity and specificity; and (2) a droplet microfluidic system that enables thousands of simultaneous, spatially multiplexed detection reactions at nanoliter volumes; and (3) a novel pre-amplification strategy that further enhances sensitivity and specificity. We demonstrate bCARMEN is capable of detecting and discriminating 52 clinically relevant bacterial species and several key antibiotic resistance genes. We further develop a proof of principle system for use with stabilized reagents and a simple workflow with optical readout using a cell phone camera, opening up the possibility of a rapid point-of-care multiplexed bacterial pathogen identification and antibiotic susceptibility testing. Significance StatementIn this paper, we use a novel primer design method combined with droplet-based CRISPR Cas13 detection to distinguish 52 clinically relevant bacterial pathogens in a single assay. We also apply the method to detect and distinguish a panel of major antibiotic resistance genes, which is of critical importance in this era of rising antibiotic resistance. Finally, we make key advances towards making our diagnostic assay deployable at the point-of-care, with a simplified emulsion-free assay process that uses mobile phone camera for detection and reduces infrastructure/skilled labor requirements.

microbiology↗

bioRxiv2020

Efficient design of maximally active and specific nucleic acid diagnostics for thousands of viruses

Diagnostics, particularly for rapidly evolving viruses, stand to benefit from a principled, measurement-driven design that harnesses machine learning and vast genomic data--yet the capability for such design has not been previously built. Here, we develop and extensively validate an approach to designing viral diagnostics that applies a learned model within a combinatorial optimization framework. Concentrating on CRISPR-based diagnostics, we screen a library of 19,209 diagnostic-target pairs and train a deep neural network that predicts, from RNA sequence alone, diagnostic signal better than contemporary techniques. Our model then makes it possible to design assays that are maximally sensitive over the spectrum of a viruss genomic variation. We introduce ADAPT (https://adapt.guide), a system for fully-automated design, and use ADAPT to design optimal diagnostics for the 1,933 vertebrate-infecting viral species within 2 hours for most species and 24 hours for all but 3. We experimentally show ADAPTs designs are sensitive and specific down to the lineage level, including against viruses that pose challenges involving genomic variation and specificity. ADAPTs designs exhibit significantly higher fluorescence and permit lower limits of detection, across a viruss entire variation, than the outputs of standard design techniques. Our model-based optimization strategy has applications broadly to viral nucleic acid diagnostics and other sequence-based technologies, and, paired with clinical validation, could enable a critically-needed, proactive resource of assays for surveilling and responding to pathogens.

genomics↗

bioRxiv2020

Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (SHERLOCK and HUDSON Integration to Navigate Epidemics), a sensitive and specific integrated diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We combine the steps of SHERLOCK into a single-step reaction and optimize HUDSON to accelerate viral inactivation in nasopharyngeal swabs and saliva. SHINEs results can be visualized with an in-tube fluorescent readout -- reducing contamination risk as amplification reaction tubes remain sealed -- and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-PCR with a sample-to-answer time of 50 minutes. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.

bioengineering↗

bioRxiv2020

Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases in real-time

Viral hemorrhagic fevers (VHFs) remain some of the most devastating human diseases, and recent outbreaks of Ebola virus disease (EVD) 1,2 and Lassa fever (LF) 3,4 highlight the urgent need for sensitive, field-deployable tests to diagnose them 5,6. Here we develop CRISPR-Cas13a-based (SHERLOCK) diagnostics targeting Ebola virus (EBOV) and Lassa virus (LASV), with both fluorescent and lateral flow readouts. We demonstrate on laboratory and clinical samples the sensitivity of these assays and the capacity of the SHERLOCK platform to handle virus-specific diagnostic challenges. Our EBOV diagnostic detects both the L and NP genes, thereby eliminating the potential for false positive results caused by the rVSV{Delta}G-ZEBOV-GP live attenuated vaccine. Our two LASV diagnostics together capture 90% of known viral diversity and demonstrate that CRISPR-RNAs (crRNAs) can be effectively multiplexed to provide greater coverage of known viral diversity. We performed safety testing to demonstrate the efficacy of our HUDSON protocol in heat-inactivating and chemically treating VHF viruses before SHERLOCK testing, eliminating the need for an extraction. We developed a user-friendly field protocol and mobile application (HandLens) to report results, facilitating SHERLOCKs use in endemic regions. Finally, we successfully deployed our tests in Sierra Leone and Nigeria in response to recent outbreaks.

molecular biology↗

bioRxiv2020

CRISPR-based COVID-19 surveillance using a genomically-comprehensive machine learning approach

The emergence and outbreak of SARS-CoV-2, the causative agent of COVID-19, has rapidly become a global concern and has highlighted the need for fast, sensitive, and specific tools to surveil circulating viruses. Here we provide assay designs and experimental resources, for use with CRISPR-based nucleic acid detection, that could be valuable for ongoing surveillance. We provide assay designs for detection of 67 viral species and subspecies, including: SARS-CoV-2, phylogenetically-related viruses, and viruses with similar clinical presentation. The designs are outputs of algorithms that we are developing for rapidly designing nucleic acid detection assays that are comprehensive across genomic diversity and predicted to be highly sensitive and specific. Of our design set, we experimentally screened 4 SARS-CoV-2 designs with a CRISPR-Cas13 detection system and then extensively tested the highest-performing SARS-CoV-2 assay. We demonstrate the sensitivity and speed of this assay using synthetic targets with fluorescent and lateral flow detection. Moreover, our provided protocol can be extended for testing the other 66 provided designs. Assay designs are available at https://adapt.sabetilab.org/.

genomics↗