CRISPR screening in combination with chromatin availability sequencing


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The New York Genome Center (NYGC) and scientists at New York University (NYU) report that they have developed a genetic screening platform that jointly captures CRISPR gene perturbations and single-cell chromatin genome-wide availability. The new platform could help researchers explore how the link between genetic changes and chromatin availability may contribute to diseases such as cancer.


The inspiration for the new platform comes from recent advances in combining CRISPR's joinable screens and single-cell RNA sequencing. The new platform retains the combined CRISPR screens but integrates them with single-cell combinatorial analysis for transpose-accessible chromatin (sciATAC). As a result? CRISPR-sciATAC.


To develop CRISPR-sciATAC, the NYGC/NYU-led team expanded on the work of sciATAC that had been done by other research groups, such as the team led by Jay Shendur, MD, Ph.D., at the University of Washington. The NYGC/NYU-led team started by using a combination of human and mouse cells to create a tagging/identification process that allowed them to separate and decipher cell nuclei, as well as capture the single-stranded RNAs needed to target CRISPR. The team also used a unique, easy-to-clean transposase that was developed in the NYGC Innovation Technology Lab.



A key technical hurdle was optimizing the experimental conditions for simultaneous capture of CRISPR RNA guidance and genome fragments for accessibility profiling while keeping the nuclear envelope of each cell intact.


Details of how CRISPR-sciATAC was developed and how the platform performs in demonstrating chromatin availability profiling appeared on April 29 in Nature Biotechnology, in an article titled " Profiling Genetic Determinants of Chromatin Availability with Scalable Single-Cell CRISPR Screens." The authors of the paper point out that they originally developed a CRISPR library to target 20 chromatin-modifying genes that are commonly mutated in a variety of cancers, including breast, colon, lung, and brain cancers.


CRISPR-sciATAC workflow with initial barcoding, core merging, and re-partitioning, followed by a second round of barcoding. "Sanjana Lab, NYGC and NYU»
CRISPR-sciATAC workflow with initial barcoding, core merging, and re-partitioning, followed by a second round of barcoding. "Sanjana Lab, NYGC and NYU»

Many of these enzymes act as tumor suppressors, and their loss leads to global changes in chromatin availability. For example, the team showed that the loss of the EZH2 gene, which encodes histone methyltransferase, led to increased gene expression in several previously silenced developmental genes.


Ultimately, the scientists applied CRISPR-sciATAC to target 105 chromatin-related genes in human myeloid leukemia cells. This allowed the scientists to generate data on chromatin availability for 30,000 single cells.


"We correlate the loss of specific chromatin reconstructors with changes in availability worldwide and at the binding sites of individual transcription factors (TF)," the authors of the new study write. "For example, we show that the loss of H3K27 EZH2 methyltransferase increases availability in heterochromatic regions involved in embryonic development and causes gene expression in HOXA and HOXD clusters. In a subset of regulatory objects, we also analyze changes in the nucleosomal interval after the loss of chromatin remodelers."


Essentially, the scientists used the programmability of the CRISPR gene editing system to knock out almost all the genes associated with chromatin in parallel. By targeting more than 100 genes associated with chromatin, the scientists were able to create a "chromatin atlas" that determines how the genome changes in response to the loss of these proteins.


The atlas shows that different subdivisions in each of the 17 chromatin remodeling target complexes may have different effects on genome availability. Surprisingly, almost all of these complexes have divisions where loss causes increased availability and other divisions with the opposite effect. In general, the greatest disruption of transcriptional binding factors of sites that are important functional elements in the genome was observed after the loss of the AT-rich interactive domain-containing protein 1A (ARID1A), a member of the BAF complex. Mutations in complex BAF proteins are estimated to be involved in one out of every five cancers.



"We have data on the availability of exposure capture of each gene associated with chromatin," said Noah Liscovitch-Brauer, Ph. D., co-author of the study and a researcher in the laboratory of Neville Sanjana, Ph. D., senior author of the study, a core member of NYGC, and an associate professor at New York University. "This provides a detailed map between each genome and how its loss affects the organization of the genome with single-cell resolution."


"Integrating chromatin availability profiling into CRISPR screens in the genome provides us with a new lens for understanding gene regulation," Sanjana added. "With CRISPR-sciATAC, we have a comprehensive understanding of how specific chromatin-altering enzymes and complexes alter the availability and organize interactions that control gene expression.


"Chromatin lays the foundation for gene expression, and here we can quickly measure the effect of various mutations on chromatin. We hope that this atlas will be a generally 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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