Tracing Dynamic Changes of DNA Methylation at Single-Cell Resolution

Mammalian DNA methylation plays an essential role in development. To date, only snapshots of different mouse and human cell types have been generated, providing a static view on DNA methylation. To enable monitoring of methylation status as it changes over time, we establish a reporter of genomic methylation (RGM) that relies on a minimal imprinted gene promoter driving a fluorescent protein. We show that insertion of RGM proximal to promoter-associated CpG islands reports the gain or loss of DNA methylation. We further utilized RGM to report endogenous methylation dynamics of non-coding regulatory elements, such as the pluripotency-specific super enhancers of Sox2 and miR290. Loci-specific DNA methylation changes and their correlation with transcription were visualized during cell-state transition following differentiation of mouse embryonic stem cells and during reprogramming of somatic cells to pluripotency. RGM will allow the investigation of dynamic methylation changes during development and disease at single-cell resolution.National Institutes of Health (U.S.) (Grant HD 045022

One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering

The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.United States. National Institutes of Health (HD 045022)United States. National Institutes of Health (R37CA084198

Technology development in mouse genetics and epigenetics

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.Cataloged from PDF version of thesis. Vita.Includes bibliographical references.The importance and significance of a model organism in biological research cannot be overstated. The mouse in particular has been very useful in understanding questions in many areas of research such as developmental biology, cancer biology, neuroscience and genetics. However, even though the methods to make transgenic mice and gene knockin and knockouts have been successful, they are very inefficient, labor intensive and costly. Therefore, in this thesis we developed a novel methodology to rapidly and efficiently modify the mouse genome. Using CRISPR/Cas9, a novel genome-engineering technology developed from bacteria, we were able to genetically modify mouse embryonic stem cells and make mice that carried genetic modification by zygotic injections. Using CRISPR/Cas9 we were able to make mice in as little as three weeks that contained multiple gene knockouts, single nucleotide modifications, GFP and mCherry reporter alleles, epitope-tagged alleles, and conditional alleles. Another interesting area of research in mouse genetics is epigenetic regulation, specifically how DNA methylation regulates development, gene expression, and cell state. Multiple studies have shown that this epigenetic modification plays an important regulatory role in these processes; however, the technology that has existed so far to investigate DNA methylation has only been able to look at snapshots of methylation patterns in fixed cell populations. In this thesis we have developed a novel technology named Reporter of Genomic Methylation (RGM), which allows for the investigation of methylation dynamics at single cell-resolution in vivo. The RGM technology was developed using a minimal synthetic secondary DMR promoter that drives the expression of a florescent protein. Using CRISPR/Cas9 the RGM reporter can be integrated into any genomic locus where it can report on the methylation state of its surroundings. We further show that the RGM reporter activity reflects the methylation state of non-coding regulatory elements such as promoters and enhancers. Furthermore, we show that the RGM technology allows for the dynamics of methylation and demethylation to be observed at these non-coding loci as cells transition between a pluripotent and differentiated state.by Chikdu Shakti Shivalila.Ph. D

One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering

Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty - 8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.National Institutes of Health (U.S.) (NIH grant R37-HD045022

Parkinson-associated risk variant in distal enhancer of α-synuclein modulates target gene expression

Genome-wide association studies (GWAS) have identified numerous genetic variants associated with complex diseases, but mechanistic insights are impeded by a lack of understanding of how specific risk variants functionally contribute to the underlying pathogenesis. It has been proposed that cis-acting effects of non-coding risk variants on gene expression are a major factor for phenotypic variation of complex traits and disease susceptibility. Recent genome-scale epigenetic studies have highlighted the enrichment of GWAS-identified variants in regulatory DNA elements of disease-relevant cell types. Furthermore, single nucleotide polymorphism (SNP)-specific changes in transcription factor binding are correlated with heritable alterations in chromatin state and considered a major mediator of sequence-dependent regulation of gene expression. Here we describe a novel strategy to functionally dissect the cis-acting effect of genetic risk variants in regulatory elements on gene expression by combining genome-wide epigenetic information with clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 genome editing in human pluripotent stem cells. By generating a genetically precisely controlled experimental system, we identify a common Parkinson’s disease associated risk variant in a non-coding distal enhancer element that regulates the expression of α-synuclein (SNCA), a key gene implicated in the pathogenesis of Parkinson’s disease. Our data suggest that the transcriptional deregulation of SNCA is associated with sequence-dependent binding of the brain-specific transcription factors EMX2 and NKX6-1. This work establishes an experimental paradigm to functionally connect genetic variation with disease-relevant phenotypes.National Institutes of Health (U.S.) (1R01NS088538-01)National Institutes of Health (U.S.) (2R01MH104610-15)Qatar National Research Fund (Grant NPRP 5-531-1-094

Chad Tillberg READ Poster

Chad Tillberg, Assistant Professor of Biology, reading Cockroaches: Ecology, Behavior, and Natural History, by William J. Bell, Louis M. Roth, and Christine A. Nalepa.https://digitalcommons.linfield.edu/libraries_read/1067/thumbnail.jp