Bidirectional epigenetic editing reveals hierarchies in gene regulation

Abstract

CRISPR perturbation methods are limited in their ability to study non-coding elements and genetic interactions. In this study, we developed a system for bidirectional epigenetic editing, called CRISPRai, in which we apply activating (CRISPRa) and repressive (CRISPRi) perturbations to two loci simultaneously in the same cell. We developed CRISPRai Perturb-seq by coupling dual perturbation gRNA detection with single-cell RNA sequencing, enabling study of pooled perturbations in a mixed single-cell population. We applied this platform to study the genetic interaction between two hematopoietic lineage transcription factors, SPI1 and GATA1, and discovered novel characteristics of their co-regulation on downstream target genes, including differences in SPI1 and GATA1 occupancy at genes that are regulated through different modes. We also studied the regulatory landscape of IL2 (interleukin-2) in Jurkat T cells, primary T cells and chimeric antigen receptor (CAR) T cells and elucidated mechanisms of enhancer-mediated IL2 gene regulation. CRISPRai facilitates investigation of context-specific genetic interactions, provides new insights into gene regulation and will enable exploration of non-coding disease-associated variants.

Comprehensive transcription factor perturbations recapitulate fibroblast transcriptional states

“…Extending these predictions across the genome will help refine future library design. Other notable advantages of CRISPRa include the simplicity of multiplexing perturbations by co-expressing guides, the low cost of synthesis compared to cDNA libraries, the preservation of natural splicing and UTR regulation, and the ability to easily pair knockdown and activation 97 .…”

HyperCas12a enables highly-multiplexed epigenome editing screens

“…Many aspects of health and disease involve both increases and decreases in gene expression. Modeling those systems via simultaneous activation of some genes and repression of others has been previously demonstrated using dCas9 orthologs and a dual sgRNA expression vector 13,18,19 . Here, we demonstrate an alternative approach using a hybrid dCas12a pre-crRNA to simultaneously activate two genes and repress two genes.…”

“…Recent work to experimentally test and validate thousands of CRE-gene pairs found between two and three CREs per gene, while predictive modeling of millions of CRE-promoter interactions estimates about four CREs per gene 11,12 . It is also common that coordinated expression of several genes controls aspects of cell differentiation and disease [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] .…”

“…Moreover, by delivering multiple crRNAs at once, it is possible to study interactions between CREs. For example, dCas9-based studies of pairwise epigenetic perturbations have revealed combinations of transcription factors that enhance neuronal differentiation 13 ; long-distance synergistic interactions between enhancers controlling MYC expression 2 ; synthetic lethality in the human genome 27 ; and hierarchies in CRE interactions 19 .…”