New York Genome Center (NYGC) and New York University (NYU) scientists report that they have developed a genetic screening platform that jointly captures CRISPR gene perturbations and single-cell chromatin accessibility genome-wide. The new platform could help researchers study how links between genetic changes and chromatin accessibility may contribute to diseases such as cancer.
Inspiration for the new platform comes from recent successes in combining pooled CRISPR screens and single-cell RNA sequencing. The new platform keeps the pooled CRISPR screens, but integrates them with a single-cell combinatorial indexing assay for transposase-accessible chromatin (sciATAC). The result? CRISPR-sciATAC.
To develop CRISPR-sciATAC, the NYGC/NYU-led team extended sciATAC work that had been carried out by other research groups, such as the team led by Jay Shendure, MD, PhD, at the University of Washington. The NYGC/NYU-led team began by using a mix of human and mouse cells to create a tagging/identification process that allowed them to split and barcode the nuclei of cells as well as capture the single-guide RNAs required for CRISPR targeting. The team also used a unique, easy-to purify transposase that was developed in the NYGC’s Innovation Technology Lab.
A key technical hurdle was optimizing experimental conditions to simultaneously capture the CRISPR guide RNAs and genome fragments for accessibility profiling while also keeping the nuclear envelope of each cell intact.
Details of how CRISPR-sciATAC was developed—and how the platform performed in a demonstration of chromatin accessibility profiling—appeared April 29 in Nature Biotechnology, in an article titled, “Profiling the genetic determinants of chromatin accessibility with scalable single-cell CRISPR screens.” The article’s authors indicate that they initially designed a CRISPR library to target 20 chromatin-modifying genes that are commonly mutated in different cancers, including breast, colon, lung, and brain cancers.
Many of these enzymes act as tumor suppressors, and their loss results in global changes in chromatin accessibility. For example, the group showed that loss of the gene EZH2, which encodes a histone methytransferase, resulted in an increase in gene expression across several previously silenced developmental genes.
Ultimately, the scientists applied CRISPR-sciATAC to target 105 chromatin-related genes in human myelogenous leukemia cells. Doing so allowed the scientists to generate chromatin accessibility data for ~30,000 single cells.
“We correlate the loss of specific chromatin remodelers with changes in accessibility globally and at the binding sites of individual transcription factors (TFs),” the authors of the new study wrote. “For example, we show that loss of the H3K27 methyltransferase EZH2 increases accessibility at heterochromatic regions involved in embryonic development and triggers expression of genes in the HOXA and HOXD clusters. At a subset of regulatory sites, we also analyze changes in nucleosome spacing following the loss of chromatin remodelers.”
Essentially, the scientists harnessed the programmability of the gene editing system CRISPR to knock out nearly all chromatin-related genes in parallel. By targeting more than 100 chromatin-related genes, the scientists managed to build a “chromatin atlas” that charts how the genome changes in response to loss of these proteins.
The atlas shows that different subunits within each of the 17 chromatin remodeling complexes targeted can have different effects on genome accessibility. Surprisingly, nearly all of these complexes have subunits where loss triggers increased accessibility and other subunits with the opposite effect. Overall, the greatest disruption in transcription factor binding sites, which are important functional elements in the genome, was observed after loss of AT-rich interactive domain-containing protein 1A (ARID1A), a member of the BAF complex. Mutations in BAF complex proteins are estimated to be involved in one out of every five cancers.
“We have accessibility data capturing the impact of every chromatin-related gene,” said Noa Liscovitch-Brauer, PhD, the study’s co-first author and a researcher in the laboratory of Neville Sanjana, PhD, the study’s senior author, a core member of the NYGC, and an assistant professor at NYU. “This provides a detailed map between each gene and how its loss impacts genome organization with single-cell resolution.”
“Integrating chromatin accessibility profiling into the genome-wide CRISPR screens provides a new lens for us to understand gene regulation,” Sanjana added. “With CRISPR-sciATAC, we have a comprehensive view into how specific chromatin-modifying enzymes and 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 chromatin rapidly. We hope this atlas will be a broadly useful resource for the community and that CRISPR-sciATAC will be used to produce similar atlases in other biological systems and disease contexts.”