Biology subjects

Liu, D. R.

Publications and source records attributed to Liu, D. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Creation of a High-Resolution, Continually Evolvable Lineage Tracer

Cellular diversity is required for the proper function of many tissues, including the retina, with its ~130 cell types.1-3 Previous lineage studies suggested that retinal progenitor cells (RPCs) generate cell diversity through biased cell divisions.4-9 However, the extent to which each progenitor cell contributes to diversity, and the overarching rules governing the lineages of the collection of progenitor cells, are unknown. While classic lineage tracing approaches including retroviral labeling4,6,10, dye injection11,12, and time-lapse microscopy13,14 were used to make the foundational insights regarding retinal lineages, they cannot be used to interrogate multigenerational lineages at scale. Here we describe the creation of an evolvable lineage tracing tool, "SCRIBBLE" for Sequential Combinatorial Recorder for Iterative Barcode-Based Lineage Evolution. SCRIBBLE uses prime editor to iteratively edit a barcode sequence while preserving the order of genesis of clonally related progeny. Through a series of rationally designed high-throughput screens and design optimizations, we identified novel sequences that support a high level of barcode editing and information content. Evaluation of SCRIBBLE in embryonic mouse retina recapitulated known lineages in retinal development, demonstrating that SCRIBBLE is capable of high-resolution lineage tracing in a complex tissue.

developmental biology↗

bioRxiv2026

In vivo HSPC gene therapy of hemoglobinopathies without drug selection of corrected cells

In vivo hematopoietic stem/progenitor cell (HSPC) gene therapy remains limited by low gene-editing efficiency and a lack of clinically applicable selection strategies to enrich therapeutically corrected progeny. We used in vivo base and prime editing to introduce a nonpathogenic EPOR variant into HSPCs, conferring erythropoietin hypersensitivity and promoting preferential expansion of gene-corrected erythroid cells. EPOR editing was combined with three therapeutic approaches for the correction of hemoglobinopathies: {gamma}-globin gene addition, {gamma}-globin reactivation, or correction of the sickle cell disease mutation. Tropism-modified helper-dependent adenoviral vectors (HDAd6/3+) targeting HSPCs were used to simultaneously deliver the EPOR-editing machinery and the corresponding therapeutic components. In vitro studies in an erythroid progenitor cell line and primary CD34+ cells demonstrated that the EPORW439* variant conferred a strong proliferative advantage to therapeutically modified erythroid progenitors. Mice humanized with CD34+ cells from a {beta}/{beta}-thalassemia patient were subjected to EPORW439*-mediated EPO hypersensitivity alongside a therapeutic {gamma}-globin transgene which resulted in >70% HbF-positive erythroid cells and substantial reversion of the disease-associated phenotype, including reduced oxidative stress, near-complete elimination of splenic iron deposition, and reduced splenomegaly. Importantly, these effects were achieved after simple intravenous administration of the vectors following HSPC mobilization and cytokine prophylaxis, without subsequent pharmacologic selection. Together, these findings establish a strategy to amplify the therapeutic benefit of otherwise limited in vivo HSPC gene editing for hemoglobinopathies.

bioengineering↗

bioRxiv2026

ESCAPE: assigning site-specific activity to covalent ligands in cells by prime editing

Chemical proteomics has identified covalent ligands targeting cysteine residues across many hundreds of human proteins. The functional effects of these liganding events, however, remain challenging to assign at scale. Here we describe ESCAPE (Endogenous Site-specific Competition Assays using Prime Editors), a platform for the site-resolved functional analysis of covalent ligands in cells. In this method, cysteine-to-serine substitutions are generated by prime editing to abrogate covalent ligand-protein interactions, and the impact of these edits on ligand-induced cellular phenotypes is quantified through allele frequency-based resistance scores. Applied to ligandable cysteines mapped by activity-based protein profiling in 50+ proteins, ESCAPE identified multiple covalent ligand-protein interactions that impair cancer cell growth, including azetidine butynamides that target a non-orthosteric cysteine in the RNA helicase DDX49 to disrupt 18S rRNA processing, 40S ribosome assembly, and protein synthesis. ESCAPE thus provides a scalable framework for the functional characterization of covalent ligands targeting structurally and mechanistically diverse proteins.

biochemistry↗

bioRxiv2026

CD117 epitope-shielded hematopoietic stem cell transplantation with toxin-free conditioning and in vivo selection ameliorates β-thalassemia model

Clinical evidence demonstrates that ex vivo gene therapy and genome engineering of hematopoietic stem and progenitor cells (HSPCs) could represent one-time cures. However, while genome editing itself has become increasingly efficient and precise, the toxic conditioning required for hematopoietic stem cell transplantation remains a major barrier to broad clinical implementation of these otherwise curative therapies. In particular, the use of busulfan for myeloablative conditioning constitutes a major safety concern. While preclinical studies established CD117 as a promising target for antigen-specific therapy, clinical translation faced setbacks balancing efficacy and safety. To overcome current limitations, we generated a new CD117-blocking monoclonal antibody (CIM058) and demonstrate its potency to block wild-type HSPCs. To enable long-term blockade of host HSPCs even after transplantation, we used prime editing to engineer CIM058-resistant human CD34+ HSPCs. When combined, CIM058 and the epitope engineered CD34+ HSPCs ameliorated disease phenotype in a {beta}-thalassemia model. Our results suggest that this approach may overcome the reliance on busulfan or other myeloablative conditioning regimens with their associated morbidities, and by enabling toxin-free conditioning and in vivo selection of edited cells, may facilitate clinical implementation of these highly valuable genetic therapies.

bioengineering↗

bioRxiv2025

Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells

Primary human myeloid cells are promising candidates for immunotherapy, yet efficient and scalable technologies for genetic engineering and screening in these cells are limited. Here we present a virus-like particle (VLP)-based toolkit that delivers diverse CRISPR genome editing modalities to human monocytes, macrophages, and dendritic cells with high efficiency while preserving viability and innate immune responsiveness. VLP-mediated delivery of ribonucleoprotein payloads supports gene knockout, base editing and epigenetic silencing, and enables site-specific integration of large DNA sequences when combined with AAV donors for homology-directed repair. Leveraging sgRNA delivery via VPX-lentivirus combined with Cas9 protein delivery via engineered virus-like particle (eVLP) treatment ("SLICeVLP"), we performed the first pooled loss-of-function screens in human macrophages. We uncovered regulators of TNF production and CD80 expression in human macrophages, converging on TNFAIP3 as a central regulator of inflammatory polarization. TNFAIP3 ablation promoted a pro-inflammatory cell state that is resistant to suppressive polarization, and augmented cytotoxicity of engineered HER2 CAR-macrophages. Taken together, this technology platform enables unbiased discovery and characterization of functional gene targets in primary human myeloid cells.

immunology↗

bioRxiv2025

Ultrasmall chemogenetic tags with group-transfer ligands

Chemogenetic tags are valuable tools for studying functions of a given protein-of-interest (POI) lacking small-molecule ligands, but most tags are too large for several POIs. Here, we report two ultrasmall chemogenetic tags (mgTag and cTag) of 36 and 50 amino acids (aa) that, to the best of our knowledge, are the smallest reported. These tags exhibit transferase-type reactivity with their ligands to append any moiety of interest to the tag. cTag utilizes an engineered C1 domain-bearing cysteine that undergoes group-transfer reaction with its ligand. Likewise, mgTag utilizes an engineered zinc-finger domain-bearing cysteine that undergoes group-transfer reaction with its molecular-glue ligand in the presence of cereblon (CRBN). We applied these tags in the context of cell signaling and proximity induction. While the fusion of HaloTag (297 aa) to the KRASG12D (188 aa) disrupted its ability to activate the growth-signaling pathway, fusion of mgTag or cTag did not. Group-transfer of BRD4 binder to tags appended to Abelson kinase (ABL) induced proximity between ABL and BRD4, resulting in the latters phosphorylation. Deletion of the transferase-type reactivity reduced phosphorylation levels, suggesting that proximity-inducing chimeras with group-transfer design may be more efficacious in certain scenarios. We envision these ultrasmall tags to have wide-ranging applications, including in basic science, biotechnology, and medicine. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/653252v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1f99888org.highwire.dtl.DTLVardef@18d3e0corg.highwire.dtl.DTLVardef@1e4a044org.highwire.dtl.DTLVardef@d610ee_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Defining the function of disease variants with CRISPR editing and multimodal single cell sequencing

Abstract / Summary ParagraphGenetic studies have identified thousands of individual disease-associated non-coding alleles, but identification of the causal alleles and their functions remain critical bottlenecks. Even though CRISPR-Cas editing has enabled targeted modification of DNA, inefficient editing leads to heterogeneous outcomes across individual cells, limiting the ability to detect functional consequences of disease alleles. To overcome these challenges, we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome, and measures cell surface protein expression. We apply this approach to investigate the effects of gene disruption, deletions in regulatory regions, and non-coding single nucleotide polymorphisms. We identify the specific effects of individual SNPs, including the state-specific effects of an IL2RA autoimmune variant in primary human T cells. Multimodal functional genomic single cell assays including DNA sequencing bridge a crucial gap in our understanding of complex human diseases by directly identifying causal variation in primary human cells.

genomics↗

bioRxiv2023

Proteome-wide base editor screens to assess phosphorylation site functionality in high-throughput

Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here, we describe "signaling-to-transcription network" mapping through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally-resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of the phosphatase PHLPP1 which promotes NFAT but inhibits NF{kappa}B activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.

systems biology↗

bioRxiv2023

Small-molecule binding and sensing with a designed protein family

Despite transformative advances in protein design with deep learning, the design of small-molecule-binding proteins and sensors for arbitrary ligands remains a grand challenge. Here we combine deep learning and physics-based methods to generate a family of proteins with diverse and designable pocket geometries, which we employ to computationally design binders for six chemically and structurally distinct small-molecule targets. Biophysical characterization of the designed binders revealed nanomolar to low micromolar binding affinities and atomic-level design accuracy. The bound ligands are exposed at one edge of the binding pocket, enabling the de novo design of chemically induced dimerization (CID) systems; we take advantage of this to create a biosensor with nanomolar sensitivity for cortisol. Our approach provides a general method to design proteins that bind and sense small molecules for a wide range of analytical, environmental, and biomedical applications.

biochemistry↗

bioRxiv2023

Self-delivering CRISPR RNAs for AAV Co-delivery and Genome Editing in vivo

Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a "protecting oligo"), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.

molecular biology↗

bioRxiv2023

A molecular glue approach to control the half-life of CRISPR-based technologies

Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (> 230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented in vivo on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latters rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors, CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor in vivo using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.

bioengineering↗

bioRxiv2023

Multiplex Base Editing to Protect from CD33-Directed Therapy: Implications for Immune and Gene Therapy

On-target toxicity to normal cells is a major safety concern with targeted immune and gene therapies. Here, we developed a base editing (BE) approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate (NHP) hematopoietic stem and progenitor cells (HSPCs) protects from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo, thus demonstrating potential for novel immunotherapies with reduced off-leukemia toxicity. For broader applications to gene therapies, we demonstrated highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in NHPs. In vitro, dual gene-edited cells could be enriched via treatment with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO). Together, our results highlight the potential of adenine base editors for improved immune and gene therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/529353v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@70c99dorg.highwire.dtl.DTLVardef@c46ef4org.highwire.dtl.DTLVardef@505fd2org.highwire.dtl.DTLVardef@c30b8c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Nanoparticle mediated in vitro and in vivo CRISPR base correction of LCA16 causing nonsense mutation rescues Kir7.1 channel function

Clinical genome editing is emerging for rare disease treatment, but one of the major limitations is the targeted delivery of CRISPR editors. We delivered base editors to the retinal pigmented epithelium (RPE) in the mouse eye using silica nanocapsules (SNC) as a treatment for retinal degeneration. Leber Congenital Amaurosis (LCA16) is a rare pediatric blindness caused by point mutations in the KCNJ13 gene, a loss-of-function inwardly rectifying potassium channel (Kir7.1) in the RPE. SNC carrying adenine base editor (ABE8e) mRNA and single-guide RNA precisely and efficiently corrected KCNJ13W53X/W53X mutation. Editing in both patient fibroblasts (47%) and human-induced pluripotent stem cell-derived RPE (LCA16-iPSC-RPE) (17%) had a negligible off-target response. Functional Kir7.1 channels were recorded from the edited LCA16-iPSC-RPE. In the LCA16 mouse model (Kcnj13W53X/+{Delta}R), RPE cells targeted SNC delivery of ABE8e mRNA preserved normal visual function measured by full-field electroretinogram (ERG). Moreover, multifocal ERG confirmed the topographic measure of electrical activity primarily originating from the edited retinal area at the injection site. Preserved retina structure, post-treatment, was established by Optical Coherence Tomography (OCT). This preclinical validation of targeted ion channel functional rescue, a challenge for pharmacological and genomic interventions, reinforces the effectiveness of nonviral genome editing therapy for rare inherited disorders. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/499808v3_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1fb405eorg.highwire.dtl.DTLVardef@3f14c1org.highwire.dtl.DTLVardef@16bb82aorg.highwire.dtl.DTLVardef@107d862_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2022

Purified cytosolic crystals from Bacillus thuringiensis as a novel active pharmaceutical ingredient (API)

Bacillus thuringiensis or Bt is a Gram-positive soil bacterium, widely and safely applied in the environment as an insecticide for combatting insect pests that damage crops and vector diseases. Dominant active ingredients made by Bt are insect-killing crystal (Cry) proteins released as crystalline inclusions upon bacterial sporulation. Some Bt Cry proteins, e.g., Cry5B, target nematodes (roundworms) and show exceptional promise as anthelmintics (cures for parasitic nematode diseases). We have recently described IBaCC (for Inactivated Bacteria with Cytosolic Crystal(s)) in which bioactive Bt Cry crystals (containing Cry5B) are fully contained within the cytosol of dead bacterial ghosts. Here we demonstrate that these IBaCC-trapped Cry5B crystals can be liberated and purified away from cellular constituents yielding Purified Cytosolic Crystals (PCC). Cry5B PCC contains [~]95% Cry5B protein out of the total protein content. Cry5B PCC is highly bioactive against parasitic nematode larvae and adults in vitro. Cry5B PCC is also highly active in vivo against experimental human hookworm and Ascaris infections in rodents. The process was scaled up to the 100 liter scale to produce PCC for a pilot study to treat two foals infected with the Ascarid, Parascaris spp. Single dose Cry5B PCC brought the fecal egg counts of both foals to zero. These studies describe the process for the scalable production of purified Bt crystals and define a new active pharmaceutical ingredient form of Bt Cry proteins. NON-TECHNICAL IMPORTANCE PARAGRAPHBacillus thuringiensis crystal proteins are widely and safely used as insecticides. Recent studies show they also can cure gastrointestinal parasitic worm (nematode) infections when ingested. However, reproducible, scalable, and practical techniques for purifying these proteins have been lacking. Here, we address this severe limitation and present scalable and practical methods for large-scale purification of potently bioactive B. thuringiensis crystals and crystal proteins. The resultant product, called Purified Cytosolic Crystals (PCC), is highly compatible with ingestible drug delivery and formulation. Furthermore, there are growing applications in agriculture and insect control where access to large quantities of purified crystal proteins are desirable and where these methods will find great utility.

microbiology↗

bioRxiv2021

Proteolysis Targeting Chimeras With Reduced Off-targets

Proteolysis Targeting Chimeras (PROTACs), a class of heterobifunctional molecules that recruit target proteins to E3 ligases, have gained traction for targeted protein degradation. However, pomalidomide, a widely used E3 ligase recruiter in PROTACs, can independently degrade other targets, such as zinc-finger (ZF) proteins, that hold key functions in normal development and disease progression. This off-target degradation of pomalidomide-based PROTACs raises concerns about their therapeutic applicability and long-term side effects. Therefore, there is a crucial need to develop rules for PROTAC design that minimize off-target degradation. In this study, we developed a high-throughput platform that interrogates the off-target degradation of ZF domains and discovered, using this platform, that PROTACs with the current design paradigm induce degradation of several ZF proteins. To identify new rules for PROTAC design, we generated a library of pomalidomide analogs that allowed systematic exploration of the impact of positional isomerism (e.g., C4 and C5 positions of the phthalimide ring), hydrogen bonding, steric and hydrophobic effects on propensities for ZF protein degradation. We found that modifications of appropriate size on the C5 position reduced off-target ZF degradation. We validated these results using immunoblotting, target engagement, and global mass spectrometric studies. We applied our newfound design principles on a previously developed ALK oncoprotein-targeting PROTAC and generated PROTACs with enhanced potency and minimal off-target degradation. We envision the reported off-target profiling platform and pomalidomide analogs will find utility in design of specific PROTACs.

pharmacology and toxicology↗

bioRxiv2021

Global-scale CRISPR gene editor specificity profiling by ONE-seq identifies population-specific, variant off-target effects

Defining off-target profiles of gene-editing nucleases and CRISPR base editors remains an important challenge for use of these technologies, therapeutic or otherwise. Existing methods can identify off-target sites induced by these gene editors on an individual genome but are not designed to account for the broad diversity of genomic sequence variation that exists within populations of humans or other organisms. Here we describe OligoNucleotide Enrichment and sequencing (ONE-seq), a novel in vitro method that leverages customizable, high-throughput DNA synthesis technology instead of purified genomic DNA (gDNA) from individual genomes to profile gene editor off-target sites. We show that ONE-seq matches or exceeds the sensitivity of existing single-genome methods for identifying bona fide CRISPR-Cas9 off-target sites in cultured human cells and in vivo in a liver-humanized mouse model. In addition, ONE-seq outperforms existing best-in-class single-genome methods for defining off-target sites of CRISPR-Cas12a nucleases, cytosine base editors (CBEs), and adenine base editors (ABEs), unveiling previously undescribed bona fide off-target sites for all these editors in human cells. Most importantly, we leveraged ONE-seq to generate the first experimentally-derived population-scale off-target profiles for Cas9 nucleases that define the impacts of sequence variants from >2,500 individual human genome sequences in the 1000 Genomes Project database. Notably, some of the variants we identified that lead to increased mutation frequencies at off-target sites are enriched in specific human populations. We validated that novel population-specific, variant-sensitive off-target sites nominated by ONE-seq in vitro can show increased frequencies of mutations in human lymphoblastoid cells (LCLs) harboring these sequence variants. Collectively, our results demonstrate that ONE-seq is a highly sensitive off-target nomination method that can uniquely be used to identify population subgroup-linked differences in off-target profiles of gene editors. ONE-seq provides an important new pathway by which to assess the impacts of global human genetic sequence diversity on the specificities of gene editors, thereby enabling a broader and more all-inclusive approach for profiling off-target effects of these transformative therapeutic technologies.

molecular biology↗

bioRxiv2020

Prime Editing in Mice Reveals the Essentiality of a Single Base in Driving Tissue-Specific Gene Expression

Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Several genome editing platforms have emerged to evaluate the functionality of TFBS in animals. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice to demonstrate the essentiality of a single TFBS, called a CArG box, in the promoter region of the Tspan2 gene. HDR-mediated substitution of three base pairs in the Tspan2 CArG box resulted in 20/37 (54%) founder mice testing positive for the correct edit. Mice homozygous for this edit showed near loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Using the same protospacer, PE2-mediated editing of a single base in the Tspan2 CArG box yielded 12/47 (26%) founder mice testing positive for the correct edit. This single base substitution resulted in [~]90% loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Targeted sequencing demonstrated all PE2 and HDR founders with some frequency of on-target editing. However, whereas no spurious on-target indels were detected in any of the PE2 founders, many HDR founders showed variable levels of on-target indels. Further, off-target analysis by targeted sequencing revealed mutations in 5/11 (45%) HDR founders but none in PE2 founders. These results demonstrate high fidelity editing of a TFBS with PE2 and suggest a new paradigm for Cre/loxP-free tissue-specific gene inactivation via single base substitution in a TFBS. The PE2 platform of genome editing represents a powerful approach for modeling and correcting relevant noncoding SNVs in the mouse.

genetics↗

bioRxiv2020

Multimodal small-molecule screening for human prion protein binders

Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP), and there are currently no therapeutic options. PrP ligands could theoretically antagonize prion formation by protecting the native protein from misfolding or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We deployed a variety of screening methods in an effort to discover binders of PrP, including 19F-observed and saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, differential scanning fluorimetry (DSF), DNA-encoded library selection, and in silico screening. A single benzimidazole compound was confirmed in concentration-response, but affinity was very weak (Kd > 1 mM), and it could not be advanced further. The exceptionally low hit rate observed here suggests that PrP is a difficult target for small-molecule binders. While orthogonal binder discovery methods could yield high affinity compounds, non-small-molecule modalities may offer independent paths forward against prion disease.

biochemistry↗