A5050901659- CRISPR and Genetic Engineering
- CAR-T cell therapy research
- Microbial Metabolic Engineering and Bioproduction
- Biofuel production and bioconversion
- Advanced biosensing and bioanalysis techniques
- Immune Cell Function and Interaction
- RNA and protein synthesis mechanisms
- RNA regulation and disease
- Enzyme Catalysis and Immobilization
- Fungal and yeast genetics research
- Viral Infectious Diseases and Gene Expression in Insects
- Cytomegalovirus and herpesvirus research
- Bacterial Genetics and Biotechnology
- Plant Virus Research Studies
- T-cell and B-cell Immunology
- Mosquito-borne diseases and control
- Hemoglobinopathies and Related Disorders
- Insect symbiosis and bacterial influences
- Biochemical and biochemical processes
- RNA Interference and Gene Delivery
- 3D Printing in Biomedical Research
- DNA Repair Mechanisms
- Catalysis for Biomass Conversion
- Epigenetics and DNA Methylation
- Evolution and Genetic Dynamics
Integrated DNA Technologies (United States)
2020-2024
Arizona State University
2017-2021
Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased efficiency to nearly 100% at all sites examined HSPCs, iPSCs, T cells, and NK cells. We show Ultra maintains high on-target specificity thereby mitigating risk for off-target making ideal complex therapeutic applications. achieved simultaneous targeting of three clinically...
Abstract CRISPR–Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing an exogenous DNA template during allows for the intentional, precise incorporation of a desired mutation via HDR pathway. However, rates often slow compared more rapid but less accurate NHEJ-mediated repair. Here, we describe...
Chimeric antigen receptor (CAR) redirected T cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR depends on retroviral transduction. With the advent of gene editing, insertion a CD19-CAR into cell (TCR) alpha constant (TRAC) locus using adeno-associated viruses transfer was demonstrated, and these showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production is complex...
Abstract Background Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma malignancies could disrupt the therapeutic landscape solid tumors. However, access to CAR-T is not adequate meet clinical needs, in part due high cost long lead times manufacturing grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 double-stranded DNA (dsDNA) or single-stranded (ssDNA) via homology-directed repair (HDR),...
Abstract Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled human and mouse epithelial cells. Here, we derive S315 as an improvement over in delivering base editor RNP. Following intratracheal aerosol delivery Cy5-labeled rhesus macaques, confirm throughout respiratory tract....
While a number of methods exist to investigate CRISPR offtarget (OT) editing, few have been compared head-to-head in primary cells after clinically relevant editing processes.Therefore, we silico tools (COSMID, CCTop, and Cas-OFFinder) empirical (CHANGE-Seq, CIRCLE-Seq, DISCOVER-Seq, GUIDE-Seq, SITE-Seq) ex vivo hematopoietic stem progenitor cell (HSPC) editing.We performed using 11 different gRNAs complexed with Cas9 protein (high-fidelity [HiFi] or wild-type versions), then targeted...
Abstract Controlling off-target editing activity is one of the central challenges in making CRISPR technology accurate and applicable medical practice. Current algorithms for analyzing do not provide statistical quantification, are sufficiently sensitive separating signal from noise experiments with low rates, address detection translocations. Here we present CRISPECTOR, a software tool that supports quantification on- genome-editing NGS data using paired treatment/control experiments. In...
Lignocellulosic biomass, especially agricultural residues, represents an important potential feedstock for microbial production of renewable fuels and chemicals. During the deconstruction hemicellulose by thermochemical processes, side products that inhibit cell growth production, such as furan aldehydes, are generated, limiting cost-effective lignocellulose conversion. Here, we developed a new approach to increase cellular tolerance expressing multidrug resistance (MDR) pumps with putative...
Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target effects, mitigation random integration non-native DNA fragments, independence vector constructions, and less regulatory restrictions. Compared to the use in animal systems, RNP-mediated is still at early development stage plants. In this study, we established an efficient simplified protoplast-based...
Abstract Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, efficacy these is hindered by use glucocorticoids, often given to patients management complications such as graft-versus-host disease. To address this limitation, we developed a novel strategy rapid generation good manufacturing practice (GMP)–grade glucocorticoid-resistant multivirus-specific (VSTs) using clustered...
The rapid development of CRISPR-Cas gene editing technologies has revolutionized genetic medicine, offering unprecedented precision and potential for treating a wide array disorders. However, assessing the risks unintended effects remains critical, is complicated by new modalities unclear analytical guidelines. We present UNCOVERseq (Unbiased Nomination CRISPR Off-target Variants using Enhanced RhPCR), an improved in cellulo off-target nomination workflow designed to sensitively nominate...
Base editors can correct disease-causing genetic variants. After a neonate had received diagnosis of severe carbamoyl-phosphate synthetase 1 deficiency, disease with an estimated 50% mortality in early infancy, we immediately began to develop customized lipid nanoparticle-delivered base-editing therapy. regulatory approval been obtained for the therapy, patient two infusions at approximately 7 and 8 months age. In weeks after initial infusion, was able receive increased amount dietary...
Abstract Recombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development function by initiating the variable (V), diversity (D), joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 range from immune dysregulation severe combined immunodeficiencies (SCIDs), causing life-threatening infections death early in life without hematopoietic transplantation (HCT). Despite...
Abstract Aromatic compounds, which are traditionally derived from petroleum feedstocks, represent a diverse class of molecules with wide range industrial and commercial applications. Significant progress has been made to alternatively sustainably produce many aromatics renewable substrates using microbial biocatalysts. While the construction both natural non‐natural pathways expanded number diversity aromatic bioproducts, pathway modularization in single‐ multi‐strain systems continues...
Cellular import of D-xylose, the second most abundant sugar in typical lignocellulosic biomass, has been evidenced to be an energy-depriving process bacterial biocatalysts. The facilitator Zymomonas mobilis, Glf, is capable importing xylose at high rates without extra energy input, but inhibited by D-glucose (the primary biomass sugar), potentially limiting utility this transporter for fermentation mixtures derived from lignocellulose. In work we developed Escherichia coli platform strain...
Abstract Efficient xylose utilization will facilitate microbial conversion of lignocellulosic sugar mixtures into valuable products. In Escherichia coli, catabolism is controlled by carbon catabolite repression (CCR). However, in E. coli such as the succinate‐producing strain KJ122 with disrupted CCR, still inhibited under fermentative conditions. To probe underlying genetic mechanisms inhibiting utilization, we evolved to enhance its fermentation abilities parallel and characterized...
Abstract The global transcriptional response of Escherichia coli to styrene and potential influence exposure source was determined by performing RNA sequencing (RNA-seq) analysis on both styrene-producing styrene-exposed cells. In cases, appears cause cell envelope DNA damage, which cells respond down-regulating key genes/pathways involved in replication, protein production, wall biogenesis. Among the most significantly up-regulated genes were those with phage shock (e.g. pspABCDE/G),...
ABSTRACT Recombination-activating genes ( RAG1 and RAG2 ) are critical in lymphoid cell development function for initiating the V(D)J-recombination process to generate polyclonal lymphocytes with broad antigen-specificity. Clinical manifestations of defective RAG1/2 range from immune dysregulation severe combined immunodeficiencies (SCID), causing life-threatening infections death early life absence hematopoietic transplantation (HCT). Haploidentical HCT without myeloablative conditioning...