A5007263489- CRISPR and Genetic Engineering
- Advanced biosensing and bioanalysis techniques
- Zebrafish Biomedical Research Applications
- RNA and protein synthesis mechanisms
- RNA Interference and Gene Delivery
- Hearing, Cochlea, Tinnitus, Genetics
- RNA modifications and cancer
- Pluripotent Stem Cells Research
- RNA Research and Splicing
- Animal Genetics and Reproduction
- Ubiquitin and proteasome pathways
- Developmental Biology and Gene Regulation
- RNA regulation and disease
- Virus-based gene therapy research
- Chromosomal and Genetic Variations
- Signaling Pathways in Disease
- Cellular transport and secretion
- Genetics, Aging, and Longevity in Model Organisms
- Peptidase Inhibition and Analysis
- Genomics and Chromatin Dynamics
- Congenital heart defects research
- Muscle Physiology and Disorders
- Glycosylation and Glycoproteins Research
- Epigenetics and DNA Methylation
- Biochemical Analysis and Sensing Techniques
Oklahoma Medical Research Foundation
2016-2025
National Human Genome Research Institute
2011-2021
National Institutes of Health
2012-2021
Umeå University
2003-2020
ID Genomics (United States)
2019
Aligarh Muslim University
2016
Govind Ballabh Pant University of Agriculture and Technology
2003
The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present high-throughput mutagenesis pipeline using technology zebrafish that will make possible both saturation the genome and large-scale phenotyping efforts. We describe cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR multiplexed, sequencing. report germline transmission data from...
The use of adeno-associated virus (AAV) as a gene therapy vector has been approved recently for clinical and demonstrated efficacy in growing number trials. However, the safety AAV challenged by single study that documented hepatocellular carcinoma (HCC) after delivery mice. Most studies have not noted genotoxicity following AAV-mediated delivery; therefore, possibility there is an association between HCC controversial. Here, we performed comprehensive large mice therapeutic delivery. Using...
Abstract Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered alleles inefficient and has only been widely used in mice. Importantly, multiplex mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation zebrafish (Danio rerio) transgenic expression both cas9 multiple single guide RNAs (sgRNAs). We describe five distinct U6 promoters sgRNA efficient biallelic inactivation tyrosinase insulin...
CRISPR/Cas9 has emerged as a versatile genome-engineering tool that relies on single guide RNA (sgRNA) and the Cas9 enzyme for genome editing. Simple, fast economical methods to generate sgRNAs have made targeted mutagenesis routine in cultured cells, mice, zebrafish other model systems. Pre-screening of target efficacy is desirable both successful minimizing wasted animal husbandry targets with poor activity. Here, we describe an easy, quick cost-effective fluorescent polymerase chain...
Transgenic methods enable the selective manipulation of neurons for functional mapping neuronal circuits. Using confocal microscopy, we have imaged cellular-level expression 109 transgenic lines in live 6 day post fertilization larvae, including 80 Gal4 enhancer trap lines, 9 Cre and 20 that express fluorescent proteins defined gene-specific patterns. Image stacks were acquired at single micron resolution, together with a broadly expressed neural marker, which used to align reporter patterns...
With the completion of zebrafish genome sequencing project, it becomes possible to analyze function genes in a systematic way. The first step such an analysis is inactivate each protein-coding gene by targeted or random mutation. Here we describe streamlined pipeline using proviral insertions coupled with high-throughput and mapping technologies widely mutagenize genome. We also report 6144 mutagenized archived F 1 's predicted carry up 3776 mutations annotated genes. Using vitro...
Substantial intrastrain variation at the nucleotide level complicates molecular and genetic studies in zebrafish, such as use of CRISPRs or morpholinos to inactivate genes. In absence robust inbred zebrafish lines, we generated NHGRI-1, a healthy fecund strain derived from founder parents sequenced depth ∼50×. Within this strain, have identified majority genome that matches reference sequence documented most variants. This has utility for many reasons, but particular it will be useful any...
Abstract Retroviruses integrate into the host genome in patterns specific to each virus. Understanding causes of these can provide insight viral integration mechanisms, pathology and evolution, is critical development safe gene therapy vectors. We generated murine leukemia virus integrations human HepG2 K562 cells subjected them second-generation sequencing, using a DNA barcoding technique that allowed us quantify independent events. characterized >3 700 000 unique events two...
G(M2) gangliosidoses are severe neurodegenerative disorders resulting from a deficiency in β-hexosaminidase A activity and lacking effective therapies. Using Sandhoff disease (SD) mouse model (Hexb(-/-)) of the gangliosidoses, we tested potential systemically delivered adeno-associated virus 9 (AAV9) expressing Hexb cDNA to correct neurological phenotype. Neonatal or adult SD normal mice were intravenously injected with AAV9-HexB -LacZ monitored for serum activity, motor function, survival....
After injury, zebrafish can restore many tissues that do not regenerate well in mammals, making it a useful vertebrate model for studying regenerative biology. We performed systematic screen to identify genes essential hair cell regeneration zebrafish, and found the heat shock protein Hspd1 (Hsp60) has critical role of cells amputated caudal fins. showed HSP60-injected extracellularly promoted proliferation both hspd1 mutant was deficient leukocyte infiltration at site injury. Topical...
Over the past three decades, a dogma has emerged regarding uniform safety and nonpathogenicity of adeno-associated virus (AAV), for excellent reasons. First, natural AAV infections in humans are common until recently had not been associated with disease. Second, recombinant (rAAV) genome remains predominantly episomal, greatly reducing risk insertional mutagenesis genotoxicity that was observed clinical trials using retroviral vectors. Third, numerous human preclinical gene delivery studies...
CRISPRz (http://research.nhgri.nih.gov/CRISPRz/) is a database of CRISPR/Cas9 target sequences that have been experimentally validated in zebrafish. Programmable RNA-guided has recently emerged as simple and efficient genome editing method various cell types organisms, including Because the technique so easy zebrafish, most valuable asset no longer mutated fish (which distribution challenges), but rather sequence to gene confirmed high mutagenic efficiency. With highly active CRISPR target,...
In the last decade, high-throughput chemical screening has become dominant approach for discovering novel compounds with therapeutic properties. Automated using in vitro or cultured cell assays have yielded thousands of candidate drugs a variety biological targets, but these approaches not resulted an increase drug discovery despite major increases expenditures. contrast, phenotype-driven screens shown much stronger success rate, which is why we developed vivo assay transgenic zebrafish...
Aminoacyl-tRNA synthetases (ARSs) are essential enzymes for faithful assignment of amino acids to their cognate tRNA. Variants in ARS genes frequently associated with clinically heterogeneous phenotypes humans and follow both autosomal dominant or recessive inheritance patterns many instances. tryptophanyl-tRNA synthetase 1 (WARS1) cause dominantly inherited distal hereditary motor neuropathy Charcot-Marie-Tooth disease. Presently, only one family biallelic WARS1 variants has been described....
Abstract Advancements in CRISPR technology, particularly the development of base editors, revolutionize genetic variant research. When combined with model organisms like zebrafish, editors significantly accelerate and refine vivo analysis variations. However, are restricted by protospacer adjacent motif (PAM) sequences specific editing windows, hindering their applicability to a broad spectrum variants. Additionally, can introduce unintended mutations often exhibit reduced efficiency living...
CRISPR base editors are crucial for precise genome manipulation. Existing APOBECbased cytosine (CBEs), while powerful, exhibit indels and sequence context limitations, where TC-context preferences restrict effective editing of CC GC motifs. To address these challenges, we evaluated various tRNA adenine deaminase (TadA)-derived CBEs, ultimately engineering zTadCBE that demonstrates high efficiency, minimized off-target effects, an expanded targeting range. Our approach integrates beneficial...
Abstract Genes and genetic variants associated with human disease are continually being discovered, but validating their causative roles mechanisms remains a significant challenge. CRISPR/Cas9 genome editing in model organisms like zebrafish can enable phenotypic characterization of founder generation (F0) knockouts (Crispants), existing approaches not amenable to high-throughput screening due high variability, cost, low phenotype penetrance. To overcome these challenges, here we provide...
Mutations in the transcription factor GATA2 cause lymphedema. is necessary for development of lymphatic valves and lymphovenous valves, patterning vessels. Here, we report that not valvular endothelial cell (VEC) differentiation. Instead, required VEC maintenance morphogenesis. also expression junction molecules VE-cadherin claudin 5 We identified miR-126 as a target GATA2, miR-126-/- embryos recapitulate phenotypes mice lacking GATA2. Primary human cells (HLECs) (HLECΔGATA2) have altered...
Abstract Regenerative medicine holds great promise for both degenerative diseases and traumatic tissue injury which represent significant challenges to the health care system. Hearing loss, affects hundreds of millions people worldwide, is caused primarily by a permanent loss mechanosensory receptors inner ear known as hair cells. This failure regenerate cells after limited mammals, while all other non-mammalian vertebrates tested were able completely these injury. To understand mechanism...