In a new resource for the scientific community, published today in Biotechnology of nature, researchers from Neville Sanjana’s lab, PhD, New York Genome Center (NYGC) and New York University (NYU) have developed CRISPR-sciATAC, a new integrative genetic screening platform that jointly captures CRISPR genetic disruptions and the accessibility of single-celled chromatin at the genome level. With this technology, they profile changes in the organization of the genome and create a large-scale atlas of the impact of the loss of chromatin-altering enzymes on the human genome. The new method exploits the programmability of the CRISPR gene-editing system to eliminate in parallel nearly all chromatin-related genes, providing researchers with more in-depth information on the role of DNA accessibility in cancer and in rare diseases involving chromatin.
Recent advances in single-cell technologies have given scientists the ability to profile chromatin, the complex of DNA and proteins that resides in the nucleus of individual cells. Chromatin is often called the “guardian” of the genome because its proteins act as packaging elements for DNA, promoting or denying access to it. This controls the processes of gene expression in the cell, such as turning specific genes on or off. Changes in the chromatin landscape have been linked to various human traits and diseases, most notably cancer.
In a first demonstration of CRISPR-sciATAC, the Sanjana Lab team designed a CRISPR library to target 20 chromatin modifier genes that are commonly mutated in different cancers, including breast, colon, lung and breast cancers. brain. Many of these enzymes act as tumor suppressors, and their loss results in overall changes in the accessibility of chromatin. For example, the group showed that loss of the EZH2 gene, which codes for a histone methytransferase, resulted in increased gene expression across several previously suppressed developmental genes.
“The CRISPR-sciATAC scale makes this dataset very unique. Here, in a uniform genetic background, we have accessibility data capturing the impact of each chromatin-bound gene. This provides a detailed map between each. gene and how its loss affects the organization of the genome, with single-cell resolution, ”said Dr. Noa Liscovitch-Brauer, postdoctoral researcher in Sanjana’s lab at the New York Genome Center and NYU and co-first author of the study .
In total, the team targeted more than 100 chromatin-related genes and developed a “chromatin atlas” that shows how the genome changes in response to the loss of these proteins. The atlas shows that different subunits within each of the 17 targeted chromatin remodeling complexes may have different effects on genome accessibility. Surprisingly, almost all of these complexes have subunits where 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 after the loss of protein 1A containing an AT-rich interactive domain (ARID1A), member of the BAF complex. BAF complex protein mutations are estimated to be involved in 1 in 5 cancers.
In addition to the CRISPR-sciATAC method, the team also developed a suite of computational methods to map the dynamic movements of nucleosomes, which are the clusters of proteins around which DNA is coiled. When there are more nucleosomes, the DNA is tightly coiled and less available to bind to transcription factors. This is exactly what the team found at specific transcription factor binding sites involved in cell proliferation after the CRISPR knockout. ARID1A. When targeting another chromatin modifying enzyme, these same sites underwent an expansion of nucleosome spacing, demonstrating the dynamics of nucleosome positioning at specific sites in the genome. The CRISPR-sciATAC method allowed the team to systematically explore this genome plasticity for multiple chromatin-modifying enzymes and transcription factor binding sites.
“We really focused on making CRISPR-sciATAC accessible – we wanted it to be something any lab could do. We produced most of the key enzymes in-house and used simple single-cell isolation methods that don’t require microfluidics or single-cell kits, ”said Dr. Antonino Montalbano, former postdoctoral fellow in the Sanjana lab at the New York Genome Center and at NYU and co-first author of the study.
To develop CRISPR-sciATAC technology, researchers used a mixture of human and mouse cells to create a labeling / identification process that allowed them to divide and encode cell nuclei as well as capture single-guide RNAs. required for CRISPR targeting. The work builds on previous ATAC-seq (sciATAC-seq) combinatorial indexing work 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-purify transposase that was developed in the NYGC Innovation Technology Lab. A key technical hurdle was optimizing the experimental conditions to simultaneously capture CRISPR guide RNAs and genome fragments for accessibility profiling while keeping the nuclear envelope of each cell intact.
“Integrating the chromatin accessibility profile into genome-wide CRISPR screens allows us to understand gene regulation,” said Dr. Sanjana, Senior Faculty, NYGC, Assistant Professor of Biology , NYU, and Assistant Professor of Neuroscience and Physiology, NYU Grossman School of Medicine, study lead author. “With CRISPR-sciATAC, we have a holistic view of how enzymes and chromatin-modifying complexes change accessibility and orchestrate the interactions that control gene expression. Chromatin sets the stage for gene expression, and here we can measure the impact of different mutations on We hope that this atlas will be a widely useful resource for the community and that CRISPR-sciATAC will be used to produce similar atlases in other biological systems and disease contexts. “
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