CRISPR-sciATAC is a novel integrative genetic screening platform that collectively records CRISPR gene disorders and the accessibility of single cell chromatin throughout the genome. The new method leverages the programmability of the CRISPR gene editing system to turn off nearly all chromatin-related genes in parallel, and offers researchers deeper insights into the role of DNA accessibility in cancer and rare diseases that involve chromatin. Photo credit: New York Genome Center’s Sanjana Lab and NYU
In a new resource for the scientific community published today in Nature Biotechnology, researchers in the laboratory of Neville Sanjana, Ph.D., at the New York Genome Center (NYGC) and New York University (NYU) developed CRISPR-sciATAC , a novel integrative genetic screening platform that collectively captures CRISPR gene disorders and the genome-wide accessibility of single cell chromatin. With this technology, they profile changes in the genome organization and create a large-scale atlas of how the loss of individual chromatin-changing enzymes affects the human genome. The new method leverages the programmability of the CRISPR gene editing system to turn off nearly all chromatin-related genes in parallel, and offers researchers deeper insights into the role of DNA accessibility in cancer and rare diseases that involve chromatin.
Recent advances in single cell technology have given scientists the ability to profile chromatin, the complex of DNA and proteins found in the nucleus of individual cells. Chromatin is often referred to as the “gatekeeper” of the genome, as its proteins act as packaging elements for DNA and either promote or deny access to it. This controls gene expression processes in the cell, such as switching certain genes on or off. Changes in the chromatin landscape have been linked to various human traits and diseases, particularly cancer.
In an initial demonstration of CRISPR-sciATAC, the Sanjana Lab team designed a CRISPR library that targets 20 chromatin-modifying genes that are commonly mutated in various cancers including breast, colon, lung and brain cancers. Many of these enzymes act as tumor suppressors and their loss leads to global changes in the accessibility of chromatin. For example, the group showed that the loss of the EZH2 gene, which codes for a histone methyltransferase, resulted in an increase in gene expression across several previously silenced developmental genes.
“The size of CRISPR-sciATAC makes this dataset very unique. Here we have accessibility data in a unified genetic background that captures the effects of each chromatin-related gene. This provides a detailed map between each gene and the effect of its loss on the Genome organization with single cell resolution, “said Dr. Noa Liscovitch-Brauer, a postdoctoral fellow in Sanjana’s lab at the New York Genome Center and NYU, and co-lead author of the study.
In total, the team targeted more than 100 chromatin-related genes and developed a “chromatin atlas” that records how the genome changes in response to the loss of these proteins. The atlas shows that different subunits in each of the 17 targeted chromatin remodeling complexes can have different effects on the accessibility of the genome. Surprisingly, almost all of these complexes have subunits in which loss triggers increased accessibility, and other subunits with the opposite effect. Overall, the greatest disruption of transcription factor binding sites, which are important functional elements in the genome, was observed following the loss of protein 1A (ARID1A) containing AT-rich interactive domains, a member of the BAF complex. It is estimated that mutations in BAF complex proteins are implicated in 1 in 5 cancers.
In addition to the CRISPR-sciATAC method, the team developed a number of computational methods to map the dynamic movements of the nucleosomes, which are the protein clusters around which the DNA is wrapped. When there are more nucleosomes, the DNA becomes tightly coiled and less available to bind transcription factors. This is exactly what the team found in specific binding sites for transcription factors involved in cell proliferation after ARID1A’s CRISPR knock-out. When a different chromatin-modifying enzyme was targeted, these nucleosome-spaced sites were expanded, demonstrating the dynamics of nucleosome positioning at specific locations in the genome. The CRISPR-sciATAC method enabled the team to systematically study this genome plasticity for multiple chromatin-modifying enzymes and transcription factor binding sites.
“We really focused on making CRISPR-sciATAC an accessible technique – we wanted it to be something any laboratory could do. We made most of the key enzymes in-house and used simple single-cell isolation methods for which none Microfluidics is required or single cell kits, “said Dr. Antonino Montalbano, a former postdoctoral fellow in Sanjana’s laboratory at the New York Genome Center and NYU and co-lead author of the study.
To develop the CRISPR-sciATAC technology, the researchers used a mixture of human and mouse cells to create a tagging / identification process that allowed them to split and barcode the nuclei, as well as those required for the CRISPR- Targeting required to capture single guide RNAs. The work builds on earlier work on the combinatorial indexing of individual cells ATAC-seq (sciATAC-seq) by Dr. Jay Shendure of the University of Washington and other groups developing new methods of single cell genomics. CRISPR-sciATAC also uses a unique, easy-to-clean transposase developed in the NYGC’s Innovation Technology Lab. An important technical hurdle was to optimize the experimental conditions to simultaneously capture the CRISPR guide RNAs and genome fragments for accessibility while keeping the nuclear envelope of each cell intact.
“The integration of chromatin accessibility profiling into the genome-wide CRISPR screens offers us a new way of understanding gene regulation,” said Dr. Sanjana, Member of Core Faculty, NYGC, Assistant Professor of Biology, NYU, and Assistant Professor of Neuroscience and Physiology. NYU Grossman School of Medicine, lead author of the study. “With CRISPR-sciATAC we have a comprehensive overview of how certain chromatin-modifying enzymes and complexes alter accessibility and control the interactions that control gene expression. Chromatin forms the basis for gene expression, and this is where we can see the influence of various mutations on it measure We hope that this atlas will be a generally useful resource for the community and that CRISPR-sciATAC will be used to generate similar atlases in other biological systems and disease contexts. ”
Chromatin transformers never rest to keep our genome open
More information:
N. Liscovitch-Brauer, A. Montalbano, J. Deng et al. Profiling the Genetic Determinants of Chromatin Accessibility with Scalable CRISPR Single Cell Screens. Nat Biotechnol (2021). doi.org/10.1038/s41587-021-00902-x
Provided by the New York Genome Center
Quote: Single-cell CRISPR technology deciphering the role of chromatin accessibility in cancer (2021, April 29), accessed on April 29, 2021 from https://phys.org/news/2021-04-single-cell-crispr- technology-deciphers-role. html
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