A5079703986- CRISPR and Genetic Engineering
- Hemoglobinopathies and Related Disorders
- RNA modifications and cancer
- RNA regulation and disease
- Prenatal Screening and Diagnostics
- RNA Research and Splicing
- Kruppel-like factors research
- Iron Metabolism and Disorders
- Pluripotent Stem Cells Research
- Genetics, Aging, and Longevity in Model Organisms
- Cancer-related gene regulation
- Epigenetics and DNA Methylation
- DNA Repair Mechanisms
- interferon and immune responses
- Erythrocyte Function and Pathophysiology
- Advanced biosensing and bioanalysis techniques
- RNA and protein synthesis mechanisms
- Plant Virus Research Studies
- Virus-based gene therapy research
- Retinal Development and Disorders
- Renal Transplantation Outcomes and Treatments
- Evolution and Genetic Dynamics
- RNA Interference and Gene Delivery
- Genetic Syndromes and Imprinting
- Pharmacological Effects and Toxicity Studies
Gladstone Institutes
2018-2025
University of California, San Francisco
2023
UNSW Sydney
2011-2022
Innovative Genomics Institute
2018-2022
University of California, Berkeley
2018-2022
XLAB (Slovenia)
2018
ETH Zurich
2018
Howard Hughes Medical Institute
2011
Dana-Farber Cancer Institute
2011
Western Sydney University
2011
Spotting off-targets from gene editing Unintended genomic modifications limit the potential therapeutic use of gene-editing tools. Available methods to find generally do not work in vivo or detect single-nucleotide changes. Three papers this issue report new for monitoring tools (see Perspective by Kempton and Qi). Wienert et al. followed recruitment a DNA repair protein breaks induced CRISPR-Cas9, enabling unbiased detection off-target cellular animal models. Zuo identified without...
Genetic disorders resulting from defects in the adult globin genes are among most common inherited diseases. Symptoms worsen birth as fetal γ-globin expression is silenced. Genome editing could permit introduction of beneficial single-nucleotide variants to ameliorate symptoms. Here, proof concept, we introduce naturally occurring Hereditary Persistance Fetal Haemoglobin (HPFH) −175T>C point mutation associated with elevated into erythroid cell lines. We show that this increases through de...
Clustered, regularly interspaced, short palindromic repeat (CRISPR)–CRISPR-associated 9 (Cas9) genome editing is revolutionizing fundamental research and has great potential for the treatment of many diseases. While immortalized cell lines become relatively easy, therapeutically relevant primary cells tissues can remain challenging. One recent advancement delivery a Cas9 protein an in vitro–transcribed (IVT) guide RNA (gRNA) as precomplexed ribonucleoprotein (RNP). This approach allows such...
Abstract Repair of double strand DNA breaks (DSBs) can result in gene disruption or modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away other outcomes. Here, we utilize a pooled CRISPR screen define host cell involvement between Cas9 DSB plasmid stranded (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor genes act repress HDR. Small molecule inhibition one these...
Naturally occurring point mutations in the
Genes encoding the human β-like hemoglobin proteins undergo a developmental switch from fetal γ-globin to adult β-globin expression around time of birth. β-hemoglobinopathies, such as sickle-cell disease and β-thalassemia, result mutations affecting gene. The only treatment options currently available carry significant adverse effects. Analyses heritable variations in (HbF) levels have provided evidence that reactivation silenced genes erythroid cells is promising therapy. repressor BCL11A...
DNA double-stranded breaks (DSBs) are especially toxic events that can be reversed by homology-directed repair (HDR), wherein information is copied from an intact template molecule. RAD51 mediates initial DSB/template pairing during homology search. A major challenge in understanding search cells the lack of tools to monitor this process. We developed proximity identification sequencing (RaPID-seq), a sensitive method marks all candidate templates searched RAD51. find HDR hierarchical, such...
Lysine methylation is widespread on human proteins, however the enzymes that catalyze its addition remain largely unknown. This limits our capacity to study function and regulation of this modification. Here we used CRISPR/Cas9 system knockout putative protein methyltransferases METTL21B METTL23 in K562 cells, determine if they methylate elongation factor eEF1A. The known eEF1A methyltransferase EEF1AKMT1 was also knocked out as a control. Targeted mass spectrometry revealed loss lysine 165...
The control of red blood cell and megakaryocyte development by the regulatory protein GATA1 is a paradigm for transcriptional regulation gene expression in lineage differentiation maturation. Most GATA1-regulated events require to bind FOG1, essentially all GATA1-activated genes are cooccupied TAL1/E2A/LMO2/LDB1 complex; however, it not known whether FOG1 simultaneously recruited GATA1. Our structural data reveal that FOG1-binding domain GATA1, N finger, can also directly contact LMO2 show...
Abstract Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform identify protein-based reagents improve HDR human hematopoietic stem and progenitor cells (HSPCs). We leverage explore sequence diversity at binding interface NHEJ...
Krüppel-like factor 3 (KLF3/BKLF), a member of the (KLF) family transcription factors, is widely expressed transcriptional repressor with diverse biological roles. Although there considerable understanding molecular mechanisms that allow KLF3 to silence activity its target genes, less known about signal transduction pathways and post-translational modifications modulate in response physiological stimuli. We observed modified range different tissues found serine/threonine kinase...
Abstract 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 powerful, sensitive assay for the unbiased identification of sites that term DISCOVER-Seq. This approach takes advantage recruitment endogenous repair factors genome-wide Cas-induced double-strand breaks. One factor, MRE11, is recruited precisely to breaks, enabling molecular characterization nuclease cut with single-base...
Krüppel-like Factor 3 (KLF3) is a broadly expressed zinc-finger transcriptional repressor with diverse biological roles. During erythropoiesis, KLF3 acts as feedback of set genes that are activated by 1 (KLF1). Noting KLF1 binds α-globin gene regulatory sequences during erythroid maturation, we sought to determine whether also interacts the locus regulate transcription. We found expression human transgenic reporter markedly up-regulated in fetal and adult cells Klf3−/− mice. Inspection mouse...
Abstract Alpha-thalassemia is an autosomal recessive disease with increasing worldwide prevalence. The molecular basis due to mutation or deletion of one more duplicated α-globin genes, and severity directly related the number allelic copies compromised. most severe form, α-thalassemia major (αTM), results from loss all four has historically resulted in fatality utero . However, transfusions now enable survival birth. Postnatally, patients face challenges similar β-thalassemia, including...
Abstract CRISPR-Cas9 genome editing is revolutionizing fundamental research and has great potential for the treatment of many diseases. While immortalized cell lines become relatively easy, therapeutically relevant primary cells tissues can remain challenging. One recent advancement delivery a Cas9 protein an in vitro transcribed (IVT) guide RNA (gRNA) as precomplexed ribonucleoprotein (RNP). This approach allows such T hematopoietic stem cells, but consequences beyond introducing foreign...