Bidirectional epigenetic editing reveals hierarchies in gene regulation

Naomi M Pacalin; Zachary Steinhart; Quanming Shi; Julia A Belk; Dmytro Dorovskyi; Katerina Kraft; Kevin R Parker; Brian R Shy; Alexander Marson; Howard Y Chang

doi:10.1038/s41587-024-02213-3

Bidirectional epigenetic editing reveals hierarchies in gene regulation

Nat Biotechnol. 2024 May 17. doi: 10.1038/s41587-024-02213-3. Online ahead of print.

Authors

Naomi M Pacalin^{1

2}, Zachary Steinhart³, Quanming Shi¹, Julia A Belk¹, Dmytro Dorovskyi^{3

4

5}, Katerina Kraft¹, Kevin R Parker^{1

6

7}, Brian R Shy^{3

5

8}, Alexander Marson^{3

4

8

9

10

11

12

13}, Howard Y Chang^{14

15}

Affiliations

¹ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, USA.
² Department of Bioengineering, Stanford University, Stanford, CA, USA.
³ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
⁴ Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
⁵ Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA.
⁶ Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA, USA.
⁷ Cartography Biosciences, Inc., South San Francisco, CA, USA.
⁸ UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
⁹ Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA.
¹⁰ Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA.
¹¹ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
¹² Diabetes Center, University of California, San Francisco, San Francisco, CA, USA.
¹³ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
¹⁴ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, USA. howchang@stanford.edu.
¹⁵ Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA. howchang@stanford.edu.

PMID: 38760566
DOI: 10.1038/s41587-024-02213-3

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.

Abstract

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