Biology subjects

Wyman, S. K.

Publications and source records attributed to Wyman, S. K..

15 recordsLinked to original sources

bioRxiv2025

Efficient in vivo mammalian neuron editing using peptide-mediated CRISPR enzyme delivery

CRISPR-mediated genome editing of the central nervous system (CNS) has the potential to revolutionize the treatment of neurological disorders, including neurodegenerative disorders such as Huntingtons disease (HD). However, the development of CRISPR therapeutics for the CNS has been hindered by challenges associated with delivery, specifically the lack of a clinically compatible, non-viral delivery technology facilitating genome editing of neurons in vivo. For most indications, two key obstacles must be overcome before therapeutic genome editing of the brain is feasible: non-toxic intracellular delivery of CRISPR cargo into neurons and establishment of strategies enabling targeted brain regions to be edited efficiently. While viral vectors have shown promise in pre-clinical models, non-viral approaches present distinct advantages: ease of manufacture as well as the transient presence of CRISPR machinery, which tempers risks of genotoxicity and immunogenicity. Peptide-enabled ribonucleoprotein (RNP) delivery of CRISPR (PERC) has emerged as a promising non-viral delivery strategy for CRISPR enzymes with initial use in primary human immune cells. In this study, we report the development of Neuro-PERC, a streamlined and optimized approach for in vivo editing of mammalian neurons. Administration of Neuro-PERC reagents via convection-enhanced delivery (CED) mediated efficient and well-tolerated neuronal genome editing. Neuro-PERC enabled robust neuronal editing in the brain of both small and large animal reporter models, and increased survival in a severe murine model of Huntingtons disease. These results establish CED-administered Neuro-PERC as a candidate delivery technology to hasten clinical translation of CRISPR-based therapies for diseases of the CNS. SummaryNeuro-PERC, a peptide-mediated CRISPR enzyme delivery technology, enables efficient in vivo mammalian neuronal editing in the brain of mice and pigs, extending survival in a murine model of Huntingtons disease when administered via convection-enhanced delivery (CED).

neuroscience↗

bioRxiv2025

Synthesis errors in oligonucleotides are propagated into the genome by homology-directed repair

Single-stranded oligonucleotides (ssODNs) are used as donor templates for therapeutic gene editing by CRISPR-Cas9 cleavage and homology-directed repair (HDR). Although ssODN sequence fidelity is critical to the safety and efficacy of editing, standard quality control methods cannot resolve individual nucleotide errors. By deep sequencing ssODNs from three manufacturers, and amplicons from edited hematopoietic stem/progenitor cells, we find that synthesis errors are present in all ssODNs tested at rates that vary more than two-fold among manufacturers, at positions that are dependent on sequence context. These synthesis errors are propagated into the genome by HDR at frequencies proportional to their abundance in the ssODN. In our sickle cell mutation correction protocol, the most prevalent SNEs are predicted to produce benign {beta}-globin variants, while the less frequent frameshift deletions will generate {beta}-thalassemia alleles. Current quality control standards are insufficient to detect these errors, and deep sequencing of ssODNs should be incorporated into regulatory submissions for clinical gene editing programs.

genomics↗

bioRxiv2024

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in, and base editing. PERC involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating the formulation with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) bearing homology-directed repair template DNA may be included. In contrast to electroporation, PERC is appealing as it requires no dedicated hardware and has less impact on cell phenotype and viability. Due to the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Here we report methods for improved PERC-mediated editing of T cells as well as novel methods for PERC-mediated editing of HSPCs, including knockout and precise knock-in. Editing efficiencies can surpass 90% using either Cas9 or Cas12a in primary T cells or HSPCs. Because PERC calls for only three readily available reagents - protein, RNA, and peptide - and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes this approach appealing for rapid deployment in research and clinical settings.

bioengineering↗

bioRxiv2024

Mitigation of Fetal Irradiation Injury from Mid-Gestation Total Body Radiation with Mitochondrial-Targeted GS-Nitroxide JP4-039

Victims of a radiation terrorist event will include pregnant women and unborn fetuses. Mitochondrial dysfunction and oxidative stress are key pathogenic factors of fetal irradiation injury. The goal of this preclinical study is to investigate the efficacy of mitigating fetal irradiation injury by maternal administration of the mitochondrial-targeted gramicidin S (GS)- nitroxide radiation mitigator, JP4-039. Pregnant female C57BL/6NTac mice received 3 Gy total body ionizing irradiation (TBI) at mid-gestation embryonic day 13.5 (E13.5). Using novel time- and-motion-resolved 4D in utero magnetic resonance imaging (4D-uMRI), we found TBI caused extensive injury to the fetal brain that included cerebral hemorrhage, loss of cerebral tissue, and hydrocephalus with excessive accumulation of cerebrospinal fluid (CSF). Histopathology of the fetal mouse brain showed broken cerebral vessels and elevated apoptosis. Further use of novel 4D Oxy-wavelet MRI capable of probing in vivo mitochondrial function in intact brain revealed significant reduction of mitochondrial function in the fetal brain after 3Gy TBI. This was validated by ex vivo Oroboros mitochondrial respirometry. Maternal administration JP4-039 one day after TBI (E14.5), which can pass through the placental barrier, significantly reduced fetal brain radiation injury and improved fetal brain mitochondrial respiration. This also preserved cerebral brain tissue integrity and reduced cerebral hemorrhage and cell death. As JP4-039 administration did not change litter sizes or fetus viability, together these findings indicate JP4-039 can be deployed as a safe and effective mitigator of fetal radiation injury from mid-gestational in utero ionizing radiation exposure. One Sentence SummaryMitochondrial-targeted gramicidin S (GS)-nitroxide JP4-039 is safe and effective radiation mitigator for mid-gestational fetal irradiation injury.

developmental biology↗

bioRxiv2021

CRISPR-Cas9-mediated knockout of CYP79D1 and CYP79D2 in cassava attenuates toxic cyanogen production

Cassava (Manihot esculenta Crantz) is a starchy root crop that supports over a billion people in tropical and subtropical regions of the world. This staple, however, produces toxic cyanogenic compounds and requires processing for safe consumption. Excessive consumption of insufficiently processed cassava, in combination with protein-poor diets, can have neurodegenerative impacts. Reducing the cyanogen content by conventional breeding is problematic due to the heterozygous nature of the crop; recombination will generally disrupt a clonally propagated cultivars suite of desirable traits. To reduce cyanide levels in cassava, we used CRISPR-mediated mutagenesis to disrupt the cytochrome P450 genes CYP79D1 and CYP79D2 whose protein products catalyze the first step in cyanogenic glucoside biosynthesis. Knockout of both genes eliminated cyanide in leaves and storage roots of cassava accession 60444 and the West African, farmer-preferred cultivar TME 419. Although knockout of CYP79D2 alone resulted in significant reduction of cyanide, mutagenesis of CYP79D1 did not, indicating these paralogs have diverged in their function. Our work demonstrates cassava genome editing for food safety, reduced processing requirements, and environmental benefits that could be readily extended to other farmer-preferred cultivars.

plant biology↗

bioRxiv2020

Identification of novel HPFH-like mutations by CRISPR base editing that elevates the expression of fetal hemoglobin

Switching hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin is an effective strategy for the treatment of beta-hemoglobinopathies. Fetal hemoglobin expression is down-regulated in the postnatal period due to the interplay of transcription regulators with the HBG promoters. However, in the hereditary persistence of fetal hemoglobin (HPFH) condition, naturally occurring point mutations in the HBG promoter causes continued expression of fetal globin even during adulthood. Inspired by this natural phenomenon, we screened the proximal promoter of human HBG genes using adenine and cytosine base editors to identify other nucleotide substitutions that could potentially lead to elevated levels of fetal globin. Both the base editors efficiently and precisely edited at the target sites with a minimal generation of indels and no deletion of one of the duplicated HBG genes. Through systematic tiling across the HBG proximal promoter, we identified multiple novel target sites that resulted in a significant increase in fetal globin levels. Further, we individually validated the top eight potential target sites from both the base editors and observed robust elevation in the fetal globin levels up to 47 %, without any detrimental effects on erythroid differentiation. Our screening strategy resulted in the identification of multiple novel point mutations and also validated the known non-deletional HPFH mutations that could elevate the fetal globin expression at therapeutically relevant levels. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter that normally mediates fetal globin silencing and identify additional targets for therapeutic upregulation of fetal hemoglobin.

genomics↗

bioRxiv2019

Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) cause the over-production of blood cells such as erythrocytes (polycythemia vera) or platelets (essential thrombocytosis). JAK2 V617F is the most prevalent somatic mutation in many MPNs, but previous modeling of this mutation in mice relied on transgenic overexpression and resulted in diverse phenotypes that were in some cases attributed to expression level. CRISPR-Cas9 engineering offers new possibilities to model and potentially cure genetically encoded disorders via precise modification of the endogenous locus in primary cells. Here we develop "scarless" Cas9-based reagents to create and reverse the JAK2 V617F mutation in an immortalized human erythroid progenitor cell line (HUDEP-2), CD34+ adult human hematopoietic stem and progenitor cells (HSPCs), and immunophenotypic long-term hematopoietic stem cells (LT-HSCs). We find no overt in vitro increase in proliferation associated with an endogenous JAK2 V617F allele, but co-culture with wild type cells unmasks a competitive growth advantage provided by the mutation. Acquisition of the V617F allele also promotes terminal differentiation of erythroid progenitors, even in the absence of hematopoietic cytokine signaling. Taken together, these data are consistent with the gradually progressive manifestation of MPNs and reveals that endogenously acquired JAK2 V617F mutations may yield more subtle phenotypes as compared to transgenic overexpression models.

cancer biology↗

bioRxiv2019

The histone chaperone FACT induces Cas9 multi-turnover behavior and modifies genome manipulation in human cells

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Using a proximity labeling system for unbiased detection of transient protein interactions in cell-free Xenopus laevis egg extract, we identified the dimeric histone chaperone FACT as an interactor of substrate-bound Cas9. Immunodepletion of FACT subunits from extract potently inhibits Cas9 unloading and converts Cas9s activity from multi-turnover to single-turnover. In human cells, depletion of FACT delays genome editing and alters the balance between indel formation and homology directed repair. Depletion of FACT also increases epigenetic marking by dCas9-based transcriptional effectors with concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.

cell biology↗

bioRxiv2018

Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair

Repair of double strand DNA breaks (DSBs) can result in gene disruption or precise gene modification via homology directed repair (HDR) from a templating donor DNA. During genome editing, altering cellular responses to DSBs may be an effective strategy to rebalance editing outcomes towards HDR and away from other repair pathways. To identify factors that regulate HDR from a double-stranded DNA donor (dsDonor), we utilized a pooled screen to define the consequences of thousands of individual gene knockdowns during Cas9-initiated HDR from a double strand plasmid donor. We find that templated dsDonor repair pathways are mostly genetically distinct from single strand donor DNA (ssDonor) repair but share aspects that include dependency upon the Fanconi Anemia (FA) pathway. We also identified several factors whose knockdown increases HDR and thus act as repressors of gene modification. Screening available small molecule inhibitors of these repressors revealed that the cell division cycle 7-related protein kinase (CDC7) inhibitor XL413 increases the efficiency of HDR by 2-3 fold in many contexts, including primary T-cells. XL413 stimulates HDR through cell cycle regulation, inducing an early S-phase cell cycle arrest that, to the best of our knowledge, is uncharacterized for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for boosting the efficiency of gene modification.

molecular biology↗

bioRxiv2018

Double Stranded DNA Damage and Genome Editing Trigger Ribosome Remodeling and Translational Shutdown

DNA damage activates a robust transcriptional stress response, but much less is known about how DNA impacts translation. The advent of genome editing via a Cas9-induced DNA double-strand break has intensified interest in understanding cellular responses to DNA damage. Here we find that DNA double-strand breaks (DSBs) induced by Cas9 or other damaging agents lead to a reduction of core ribosomal proteins, RPS27A and RPL40, and that the loss of these proteins is post-transcriptional and p53-independent. DSBs furthermore lead to the shutdown of translation through phosphorylation of eukaryotic initiation factor 2 alpha, and altering these signals affects genome editing outcomes. This DSB translational response is widespread and precedes the transcriptional response. Our results demonstrate that even a single double-strand break can lead to ribosome remodeling and reduced translational output, and suggest caution in interpreting cellular phenotypes measured immediately after genome editing.

molecular biology↗

bioRxiv2018

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach takes advantage of the recruitment of endogenous DNA repair factors for genome-wide identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides an unprecedented view of events that precede repair of the affected sites.

molecular biology↗

bioRxiv2018

In vivo selection for corrected β-globin alleles after CRISPR/Cas9 editing in human sickle hematopoietic stem cells enhances therapeutic potential

Sickle Cell Disease (SCD), one of the worlds most common genetic disorders, causes anemia and progressive multiorgan damage that typically shortens lifespan by decades; currently there is no broadly applicable curative therapy. Here we show that Cas9 RNP-mediated gene editing with an ssDNA oligonucleotide donor yields markerless correction of the sickle mutation in more than 30% of long-term engrafting human hematopoietic stem cells (HSCs), using a selection-free protocol that is directly applicable to a clinical setting. We further find that in vivo erythroid differentiation markedly enriches for corrected {beta}-globin alleles. Adoption of a high-fidelity Cas9 variant demonstrates that this approach can yield efficient editing with almost no off-target events. These findings indicate that the sickle mutation can be corrected in human HSCs at curative levels with a streamlined protocol that is ready to be translated into a therapy.\n\nONE SENTENCE SUMMARYCas9-mediated correction of the sickle mutation in human hematopoietic stem cells can be accomplished at curative levels.

genetics↗

bioRxiv2018

Controlled cycling and quiescence enables homology directed repair in engraftment-enriched adult hematopoietic stem and progenitor cells

Hematopoietic stem cells (HSCs) are the source of all blood components, and genetic defects in these cells are causative of disorders ranging from severe combined immunodeficiency to sickle cell disease. However, genome editing of long-term repopulating HSCs to correct mutated alleles has been challenging. HSCs have the ability to either be quiescent or cycle, with the former linked to stemness and the latter involved in differentiation. Here we investigate the link between cell cycle status and genome editing outcomes at the causative codon for sickle cell disease in adult human CD34+ hematopoietic stem and progenitor cells (HSPCs). We show that quiescent HSPCs that are immunophenotypically enriched for engrafting stem cells predominantly repair Cas9-induced double strand breaks (DSBs) through an error-prone non-homologous end-joining (NHEJ) pathway and exhibit almost no homology directed repair (HDR). By contrast, non-quiescent cycling stem-enriched cells repair Cas9 DSBs through both error-prone NHEJ and fidelitous HDR. Pre-treating bulk CD34+ HSPCs with a combination of mTOR and GSK-3 inhibitors to induce quiescence results in complete loss of HDR in all cell subtypes. We used these compounds, which were initially developed to maintain HSCs in culture, to create a new strategy for editing adult human HSCs. CD34+ HSPCs are edited, allowed to briefly cycle to accumulate HDR alleles, and then placed back in quiescence to maintain stemness, resulting in 6-fold increase in HDR/NHEJ ratio in quiescent, stem-enriched cells. Our results reveal the fundamental tension between quiescence and editing in human HSPCs and suggests strategies to manipulate HSCs during therapeutic genome editing.

cell biology↗

bioRxiv2018

Oligogenic inheritance of congenital heart disease involving a NKX2-5 modifier

Complex genetic inheritance is thought to underlie many human diseases, yet experimental proof of this model has been elusive. Here, we show that a human congenital heart defect, left ventricular non-compaction (LVNC), can be caused by a combination of rare, inherited heterozygous missense single nucleotide variants. Whole exome sequencing of a nuclear family revealed novel single nucleotide variants of MYH7 and MKL2 in an asymptomatic father while the offspring with severe childhood-onset LVNC harbored an additional missense variant in the cardiac transcription factor, NKX2-5, inherited from an unaffected mother. Mice bred to compound heterozygosity for the orthologous missense variants in Myh7 and Mkl2 had mild cardiac pathology; the additional inheritance of the Nkx2-5 variant yielded a more severe LVNC-like phenotype in triple compound heterozygotes. RNA sequencing identified genes associated with endothelial and myocardial development that were dysregulated in hearts from triple heterozygote mice and human induced pluripotent stem cell-derived cardiomyocytes harboring the three variants, with evidence for NKX2-5s contribution as a modifier on the molecular level. These studies demonstrate that the deployment of efficient gene editing tools can provide experimental evidence for complex inheritance of human disease.\n\nOne sentence summaryA combination of three inherited heterozygous missense single nucleotide variants underlying a familial congenital heart defect.

genetics↗

bioRxiv2017

A most wanted list of conserved protein families with no known domains

The number and proportion of genes with no known function are growing rapidly. To quantify this phenomenon and provide criteria for prioritizing genes for functional characterization, we developed a bioinformatics pipeline that identifies robustly defined protein families with no annotated domains, ranks these with respect to phylogenetic breadth, and identifies them in metagenomics data. We applied this approach to 271 965 protein families from the SFams database and discovered many with no functional annotation, including >118 000 families lacking any known protein domain. From these, we prioritized 6 668 conserved protein families with at least three sequences from organisms in at least two distinct classes. These Function Unknown Families (FUnkFams) are present in Tara Oceans Expedition and Human Microbiome Project metagenomes, with distributions associated with sampling environment. Our findings highlight the extent of functional novelty in sequence databases and establish an approach for creating a \"most wanted\" list of genes to characterize.

genomics↗