Have you been struggling to agree on a plan of action for your recent epigenetic experiments? Have you been fighting over just what strategy to use to decipher how genetic alterations affect the epigenetic landscape? While we should all follow John and Yoko’s example and try to give peace a chance, you sometimes need to go on the “ATAC” in the realm of epigenetics research!
Specifically, two bands of seasoned campaigners and top strategists recently devised uncompromising new techniques – CRISPR-sciATAC and Spear-ATAC – that combine CRISPR and chromatin accessibility assays in single cells at high-throughput to dissect how alterations to single chromatin modifiers and crucial transcription factors (TFs) impact the underlying chromatin landscape. Notably, these techniques up the throughput and ease of use compared to the previously described pioneering Pertub-ATAC methodology that cleared that way for these new advances.
So, what are these plucky new techniques, and just what can they do for the hand-to-hand “combat” that we call epigenetic research?
CRISPR-sciATAC Links Gene Alterations and Single-cell Chromatin Accessibility
Round one of this ATAC-minded article involves martial artists from a dojo led by Neville E. Sanjana (New York Genome Center/New York University), who developed a novel single-cell combinatorial indexing assay for transposase-accessible chromatin (sciATAC) protocol by adding CRISPR to the mix. Their recently unredacted battle plans describe CRISPR-sciATAC as a brash and brave technique that simultaneously captures single-cell gene perturbations and chromatin accessibility profiles at high-throughput to explore how gene mutations may impact chromatin states and gene regulation.
Let’s hear more about CRISPR-sciATAC in this rough and tumble new study from Liscovitch-Brauer and fellow epigenetic brawlers:
- First evaluations targeted over 20 chromatin modifier genes for loss-of-function perturbation with guide RNAs in Cas9-expressing human leukemia cells
- Loss of specific chromatin remodelers alters accessibility at the global scale and individual TF binding sites (TFBS)
- The assessment of scalability focused on 17 human chromatin remodeling complexes, comprising between 2 and 14 subunits each
- While the loss of the entire CoREST or NURF complexes increases accessibility and the loss of the CREST-BRG1 or SWI/SNF-pBAF complexes decreases accessibility, the loss of individual subunits prompts heterogeneous changes to chromatin accessibility
- Notably, changes in accessibility occur at enhancers to a greater degree than promoters
- Finally, a newly developed computational framework measured changes in nucleosome position in CRISPR-sciATAC (which can impact gene expression) at seven TFBS that have symmetric positioning of nucleosomes
- Loss of chromatin remodelers results in nucleosome expansion around TFBSs
- Changes to nucleosome peak positions occur at enhancers or promoters, depending on the specific TFBS
Overall, this wealth of data from just 30,000 cells suggest that CRISPR-sciATAC will fight for your right to know just how genetic perturbations affect the chromatin landscape in normal and disease states, an important goal given the link between tumorigenesis and chromatin modifier gene mutations/altered chromatin states.
“Integrating chromatin accessibility profiling into the genome-wide CRISPR screens provides a new lens for us to understand gene regulation,” said Dr. Sanjana, the study’s senior author. “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.”
Spear-ATAC Wields CRISPRi to Reveal Cancer Regulatory Networks
In round two of this single-cell CRISPR skirmish, researchers tirelessly trained by William J. Greenleaf (Stanford University School of Medicine, Stanford, CA, USA) sought to evaluate how changing TF levels via CRISPR interference (CRISPRi) affects chromatin accessibility in high-throughput with a fearsome sounding new droplet-based (10X genomics) protocol they call Spear-ATAC, or “single-cell perturbations with an accessibility read-out using scATAC-seq.”
Let’s tag in the team of Pierce and colleagues and hear how they “spear”-headed this new “ATAC” on single cells to discover how changing TF levels affects epigenetic responses and TFBS accessibility:
- Validation efforts for Spear-ATAC targeted the silencing of the hematopoietic differentiation and development-associated TF GATA1 through a dCas9-KRAB repressor fusion protein guided to specific sites via guide RNAs
- GATA1 knockdown decreases accessibility at GATA motifs, known GATA1 binding sites, the GATA1 locus (as expected), and erythroid-specific genes but increases accessibility at megakaryocyte-specific genes
- Time course studies of chromatin accessibility dynamics after TF knockdown targeted 6 TFs for knockdown with Spear-ATAC and evaluated responses over 21 days
- While local accessibility near erythroid and megakaryocytic genes alters as a function of time following GATA1 knockdown, other sites (such as STAT5 motifs) display a more complex response, emphasizing how timing influences chromatin accessibility findings following gene silencing
- The evaluation of Spear-ATAC high-throughput analytic capacity targeted 36 TFs
- GATA1, NFE2, KLF1, FOSL1, and NRF1 knockdown prompts robust motif accessibility differences that may be used to establish regulatory relationships
The authors note a wealth of additional exciting applications of Spear-ATAC, including the creation of TF interaction maps following multiplexed perturbations, the evaluation of how disease-related mutations alter TF occupancy (when combined with high-fidelity editing methods), and the delineation of relationships between cells during differentiation or tumorigenesis.
CRISPR and ATAC – The Perfect Strategy for Single-cell High-throughput Analysis?
The confrontation of CRISPR-sciATAC and Spear-ATAC may go down as one of the greatest strategic one-on-ones in epigenetic history, and the fallout could result in a much deeper understanding of how chromatin modifiers and TFs alter the chromatin landscape at the single-cell level. Can going on the “ATAC” using these two new techniques tell us more regarding human development, disease development, and aging in the future?