Feeling perturbed by the limitations of current chromatin assays? Looking for something high throughput that combines multiplexed genome editing with single cell resolution of open chromatin? Well look no further than Perturb-ATAC. The lab of Paul Khavari (Stanford, California) sought to address the above challenges with simultaneous measurements of perturbations and chromatin state in single cells.
The talented team has been interested in assessing chromatin accessibility for some time using ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing). ATAC-seq makes use of a hyperactive mutant Tn5 transposase that can simultaneously fragment and tag DNA in open chromatin with a sequencing adapter. Then, adapter-ligated fragments are isolated and sequenced. In Perturb-ATAC, Cas9+ cells are first exposed to a lentiviral sqRNA library to knock down/out a set of regions of interest. Cells are then captured in single-cell chambers where, transposition, barcode amplification, and whole-genome PCR amplification occur. The sgRNAs targeting each specific cell are identified by interpreting the sequencing data.
As proof of principle, they applied the method to study several trans-regulatory factors including transcription factors, chromatin modifiers, and human and viral ncRNAs in primary cells and cell lines. In total they assessed 63 genotypes using about 4,300 single cells. Here’s what they found:
- The Perturb-ATAC output allows for the assessment of individual cis-regulatory elements, inference of TF activity from cis-regulatory modules, and nucleosome positioning and occupancy all from single cells
- Primary human epidermal cells have three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs reveals epistatic relationships
Based on their data, the authors estimate that 20 cells/CRIPSPR genotype will be ideal for use of this technology going forward. As microfluidic cell sorting technologies improve, this could allow for assessment of hundreds or thousands of CRISPR genotypes in one ATAC-seq experiment. The authors expect that this technique will particularly useful for dissecting gene regulatory networks in development and disease.
So why continue to be perturbed by other epigenomic techniques? Give Perturb-ATAC a try in Cell, January 2019