Doctor of Philosophy, Weizmann Institute Of Science (2010)
Matthew Porteus, Postdoctoral Faculty Sponsor
Expanded CAG/CTG repeat tracts are the genetic basis for more than a dozen inherited neurological disorders including Huntingtons disease, myotonic dystrophy, and several spinocerebellar ataxias. Despite the multitude of pathologies underlying these disorders, they all share common etiology: the expansion of CAG/CTG repeats.
In collaboration with the Mittelman lab at Virginia Tech, we made engineered nucleases that target this common feature, expanded repeats, in human patient cells to induce repeat contractions. We have developed an innovative high-throughput sequencing assay based on the Pacific Bioscience technology for sequencing expanded repeats and will use this assay to measure the effectiveness of our approach.
Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a method for quantifying individual genome-editing outcomes at any site of interest with single-molecule real-time (SMRT) DNA sequencing. We show that this approach can be applied at various loci using multiple engineered nuclease platforms, including transcription-activator-like effector nucleases (TALENs), RNA-guided endonucleases (CRISPR/Cas9), and zinc finger nucleases (ZFNs), and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach offers a technique for studying double-strand break repair, facilitates the evaluation of gene-editing technologies, and permits sensitive quantification of editing outcomes in almost every experimental system used.
View details for DOI 10.1016/j.celrep.2014.02.040
View details for Web of Science ID 000334298200028
View details for PubMedID 24685129
Tal-effector nucleases (TALENs) are engineered proteins that can stimulate precise genome editing through specific DNA double-strand breaks. Sickle cell disease and β-thalassemia are common genetic disorders caused by mutations in β-globin, and we engineered a pair of highly active TALENs that induce modification of 54% of human β-globin alleles near the site of the sickle mutation. These TALENS stimulate targeted integration of therapeutic, full-length beta-globin cDNA to the endogenous β-globin locus in 19% of cells prior to selection as quantified by single molecule real-time sequencing. We also developed highly active TALENs to human γ-globin, a pharmacologic target in sickle cell disease therapy. Using the β-globin and γ-globin TALENs, we generated cell lines that express GFP under the control of the endogenous β-globin promoter and tdTomato under the control of the endogenous γ-globin promoter. With these fluorescent reporter cell lines, we screened a library of small molecule compounds for their differential effect on the transcriptional activity of the endogenous β- and γ-globin genes and identified several that preferentially upregulate γ-globin expression.
View details for DOI 10.1093/nar/gkt947
View details for Web of Science ID 000331138100059