Chromatin modifications are involved in all basic DNA-templated processes in human cells including transcription, and are mis-regulated in many diseases. With CRISPR-targeting techniques, we can write particular chromatin modifications and then measure how gene expression changes in response. This will enable us to understand how gene expression changes in disease states and how to rationally design therapeutics to reverse those changes. One of the challenges to using CRISPR perturbations to study non-coding regulatory elements is CRISPR off- target activity. Here, we show that off-target effects in non-coding perturbation experiments can be associated with significant toxicity in human cells, not only with DNA-cleaving Cas9, but also with epigenome-modifying CRISPRi/a tools. After removing off-target-prone guide RNAs, we can use CRISPR to accurately link non-coding regulatory elements with genes. These perturbation experiments are critical to learn the causal functions of chromatin modifications at specific genomic elements. However, most experiments to date have used CRISPRi, with one particular KRAB domain that establishes one particular heterochromatin state. Existing tools to manipulate chromatin state are largely drawn from a small fraction of the thousands of natural chromatin regulatory complexes; most suffer from partial or transient effects, and exhibit high variability across loci and cell types. A more complete toolbox of compact, efficient domains for pathway-specific chromatin perturbations will transform our ability to determine the causal function of particular chromatin modifications across the human genome. Here, we propose to systematically measure the gene expression effects of recruiting chromatin regulator protein domains to a promoter. This is made possible by our recent development of a high-throughput chromatin regulator recruitment assay in human cells, capable of measuring activity for tens of thousands of regulator domains simultaneously. Using this system, we will recruit-and-release CR variants from the promoter, and then measure the magnitude and permanence of transcriptional silencing at a reporter locus. We will then use epigenomic mapping assays to determine the chromatin modifications that underpin the silencing functions of these novel chromatin regulators. After characterizing thousands of domains drawn from all the different chromatin regulatory complexes, we will create and share a detailed resource of compact and efficient domains that can be fused onto CRISPR DNA- binding proteins in order to recruit desired chromatin regulatory complexes to act upon a genomic element. In order to positively impact human health with these approaches, I propose to leverage this training to develop new methods that dissect transcriptional dysregulation in kidney disease during the postdoctoral phase.
Transcriptional dysregulation is common characteristic of cells in disease states, but the combinatorial complexity with which non-coding elements and chromatin regulatory proteins interact to determine gene expression makes it difficult to determine how transcriptional dysregulation arises and how it can be therapeutically reversed. In the F99 phase, I improve high- throughput CRISPR methods to accurately link regulatory elements with genes and I develop high-throughput recruitment assays to systematically identify the domains within human chromatin regulators that repress gene expression. In the K00 phase, I propose to develop high- throughput approaches that dissect transcriptional dysregulation in kidney disease.
