Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142, USA. Electronic address:
Published: April 2021
AI Article Synopsis
Article Abstract
A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.
Background: Asthma is a global chronic respiratory disease with complex pathogenesis. While current therapies offer some relief, they often fall short in effectively managing symptoms and preventing exacerbations for numerous patients. Thus, understanding its mechanisms and discovering new drug targets remains a pressing need for better treatment.
Heat shock transcription factors (Hsfs) play important roles in plant developmental regulations and various abiotic stress responses. However, their evolutionary mechanism of freezing tolerance remains poorly understood. In our previous transcriptomics study based on DNA methylation sequencing, the BnaHsfA2 was found to be significantly accumulated in winter rapeseed (Brassica rapa L.
Single-cell methods to assess DNA methylation have not achieved the same level of cell throughput per experiment compared to other modalities, with large-scale datasets requiring extensive automation, time, and other resources. Here, we describe sciMETv3, a combinatorial indexing-based technique that enables atlas-scale libraries to be produced in a single experiment. To reduce the sequencing burden, we demonstrate the compatibility of sciMETv3 with capture techniques to enrich regulatory regions, as well as the ability to leverage enzymatic conversion, which can yield higher library diversity.
Factors influencing variance of DNA methylation in vegetatively reproducing plants, both terrestrial plants and aquatic seagrasses, is just beginning to be understood. Improving our knowledge of these mechanisms will increase understanding of transgenerational epigenetics in plant clones, of the relationship between DNA methylation and seagrass development, and of the drivers of epigenetic variation, which may underly acclimation in clonally reproducing plants. Here, we sampled leaves, rhizomes and roots of three physically and spatially separated ramet sections from a clonally propagated field of the seagrass Zostera marina.
