A5022701292- CRISPR and Genetic Engineering
- Advanced biosensing and bioanalysis techniques
- RNA Interference and Gene Delivery
- CAR-T cell therapy research
- Virus-based gene therapy research
- Hemoglobinopathies and Related Disorders
- Viral Infectious Diseases and Gene Expression in Insects
- Mosquito-borne diseases and control
- RNA regulation and disease
- Immune Cell Function and Interaction
- bioluminescence and chemiluminescence research
- T-cell and B-cell Immunology
- Innovation and Socioeconomic Development
- RNA and protein synthesis mechanisms
- Cytomegalovirus and herpesvirus research
- MicroRNA in disease regulation
- Plant Virus Research Studies
- Biosensors and Analytical Detection
- Epigenetics and DNA Methylation
- Evolution and Genetic Dynamics
- DNA Repair Mechanisms
- Animal Genetics and Reproduction
- RNA Research and Splicing
- Genetics, Aging, and Longevity in Model Organisms
- Biomedical Ethics and Regulation
Integrated DNA Technologies (United States)
2011-2024
University of Iowa
2006-2009
RNA interference (RNAi) has been extensively employed for in vivo research since its use was first demonstrated mammalian cells 10 years ago. Design rules have improved, and it is now routinely possible to obtain reagents that suppress expression of any gene desired. At the same time, increased understanding molecular basis unwanted side effects led development chemical modification strategies mitigate these concerns. Delivery remains single greatest hurdle widespread adoption RNAi methods....
Genome editing using the CRISPR/Cas9 system requires presence of guide RNAs bound to Cas9 endonuclease as a ribonucleoprotein (RNP) complex in cells, which cleaves host cell genome at sites specified by RNAs. New genetic material may be introduced during repair double-stranded break via homology dependent (HDR) if suitable DNA templates are delivered with CRISPR components. Early methods used plasmid or viral vectors make these components cell, however newer approaches recombinant protein...
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...
A point mutation in sickle cell disease (SCD) alters one amino acid the β-globin subunit of hemoglobin, with resultant anemia and multiorgan damage that typically shortens lifespan by decades. Because SCD is caused a single mutation, hematopoietic stem cells (HSCs) can be harvested, manipulated, returned to an individual, it attractive target for gene correction.An optimized Cas9 ribonucleoprotein (RNP) ssDNA oligonucleotide donor together generated correction at least allele more than 30%...
Genome editing of human cluster differentiation 34+ (CD34+) hematopoietic stem and progenitor cells (HSPCs) holds great therapeutic potential. This study aimed to optimize on-target, ex vivo genome using the CRISPR-Cas9 system in CD34+ HSPCs create a clear workflow for precise identification off-target effects. Modified synthetic guide RNAs (gRNAs), either 2-part gRNA or single-guide RNA (sgRNA), were delivered as part ribonucleoprotein (RNP) complexes, targeting therapeutically relevant...
Abstract T-cell malignancies are associated with frequent relapse and high morbidity, which is partly due to the lack of effective or targeted treatment options. To broaden use CAR-T cells in pan malignancies, we developed an allogeneic “universal” CD2-targeting cell (UCART2), CD2 antigen deleted prevent fratricide, receptor removed GvHD. UCART2 demonstrated efficacy against T-ALL CTCL prolonged survival tumor-engrafted NSG mice vivo. evaluate impact on function, generated CD19 (UCART19)...
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...
Despite progress in identifying molecular drivers of cancer, it has been difficult to translate this knowledge into new therapies, because many the causal proteins cannot be inhibited by conventional small molecule therapeutics. RNA interference (RNAi), which uses RNAs inhibit gene expression, provides a promising alternative reach traditionally undruggable protein targets shutting off their expression at messenger (mRNA) level. Challenges for realizing potential RNAi have included...
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...
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...
PEGylated glycoproteins (PGPs) were synthesized by copolymerizing a Cys-terminated PEG−peptide, glycopeptide, and melittin peptide. Compositionally unique PGPs prepared varying the ratio of PEG−peptide (20−90%) (0−70%) with constant amount glycopeptide (10%). The purified RP-HPLC, characterized for molecular weight polydispersity GPC-HPLC SDS-PAGE composition RP-HPLC following reduction to form monomeric peptides. formed DNA condensates 200−300 nm in diameter that administered mice via tail...
ABSTRACT Sickle Cell Disease (SCD), one of the world’s 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 Cas9 RNP-mediated gene editing with an ssDNA oligonucleotide donor yields markerless correction sickle mutation in more than 30% long-term engrafting human hematopoietic stem cells (HSCs), using a selection-free protocol directly to clinical...