Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

Li, Kailong; Liu, Xin; Cao, Hui; Zhang, Yuannyu; Gu, Zhimin; Liu, Xin; Yu, Andy; Kaphle, Pranita; Dickerson, Kathryn E.; Ni, Min; Xu, Jian

doi:10.1038/s41467-020-14362-5

Cited by 150 publications

(133 citation statements)

References 72 publications

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“…Such strategies enable an unprecedented ability to investigate larger transcriptional networks rather than relying on studies of individual genes. Likewise, recent studies have devoted additional attention to modulation of non-coding RNA and cis-regulatory element function using dCas9 strategies 73,74 . The adapted CRISPRi dCas9-KRAB-MeCP2 system described here is compatible with these approaches and can easily be harnessed for expanded gene silencing via the use of multiplexed sgRNA expression cassettes, targeting non-coding RNA loci, or trafficking the system to regulatory regions of interest.…”

Section: Discussionmentioning

confidence: 99%

An improved CRISPR/dCas9 interference tool for neuronal gene suppression

Duke

Revanna

Sultan

et al. 2020

Preprint

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The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective promoter human synapsin 1 enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.Correspondence to Jeremy Day (jjday@uab.edu | day-lab.org | @DayLabUAB)

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Section: Discussionmentioning

confidence: 99%

An improved CRISPR/dCas9 interference tool for neuronal gene suppression

Duke

Revanna

Sultan

et al. 2020

Preprint

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“…MCP tags are then fused to effector domains that are co-transfected with dCas9 and gRNAs. With these modifications, dCas9-gRNA complexes can now recruit a greater number of repressive units to Page 31 of 71 augment transcriptional repression (Li et al 2020). This method, termed enhancer-targeting CRISPR interference (enCRISPRi), coupled both KRAB and LSD1 together by fusing KRAB to dCas9 (dCas9-KRAB) and recruiting MCP-fused LSD1 (MCP-LSD1) to gRNA-MS2 ( Figure 3H) (Li et al 2020).…”

Section: Crispr Interferencementioning

confidence: 99%

“…With these modifications, dCas9-gRNA complexes can now recruit a greater number of repressive units to Page 31 of 71 augment transcriptional repression (Li et al 2020). This method, termed enhancer-targeting CRISPR interference (enCRISPRi), coupled both KRAB and LSD1 together by fusing KRAB to dCas9 (dCas9-KRAB) and recruiting MCP-fused LSD1 (MCP-LSD1) to gRNA-MS2 ( Figure 3H) (Li et al 2020). This compound approach displayed stronger gene repression compared to either domain alone when targeted to the β-globin LCR (Li et al 2020).…”

Section: Crispr Interferencementioning

confidence: 99%

“…This method, termed enhancer-targeting CRISPR interference (enCRISPRi), coupled both KRAB and LSD1 together by fusing KRAB to dCas9 (dCas9-KRAB) and recruiting MCP-fused LSD1 (MCP-LSD1) to gRNA-MS2 ( Figure 3H) (Li et al 2020). This compound approach displayed stronger gene repression compared to either domain alone when targeted to the β-globin LCR (Li et al 2020). Furthermore, enCRISPRi has been deployed in mouse studies with the knock-in and recruitment of dCas9-KRAB and MCP-LSD1 to hematopoietic stem cells, demonstrating the feasibility of targeting either individual or multiple enhancers for the validation of essential regulatory elements required for hematopoiesis (Li et al 2020).…”

Section: Crispr Interferencementioning

confidence: 99%

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Transcriptional enhancers: from prediction to functional assessment on a genome-wide scale

Tobias

Abatti

Moorthy

et al. 2021

Genome

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Enhancers are cis-regulatory sequences located distally to target genes. These sequences consolidate developmental and environmental cues to coordinate gene expression in a tissue-specific manner. Enhancer function and tissue specificity depend on the expressed set of transcription factors, which recognize binding sites and recruit cofactors that regulate local chromatin organization and gene transcription. Unlike other genomic elements, enhancers are challenging to identify because they function independently of orientation, are often distant from their promoters, have poorly defined boundaries and display no reading frame. In addition, there are no defined genetic or epigenetic features that are unambiguously associated with enhancer activity. Over recent years there have been developments in both empirical assays and computational methods for enhancer prediction. We review genome-wide tools, CRISPR advancements and high-throughput screening approaches that have improved our ability to both observe and manipulate enhancers in vitro at the level of primary genetic sequences, chromatin states and spatial interactions. We also highlight contemporary animal models and their importance to enhancer validation. Together, these experimental systems and techniques complement one another and broaden our understanding of enhancer function in development, evolution, and disease.

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“…Activator and repressor systems can be combined with histone code epi-editors resulting in an extension of their ability to activate or repress target genes not only through recruitment of transcription machinery on the promoter but also affecting cis-regulatory elements [82].…”

Section: Gene Regulation By Crispra and Crispri Systemsmentioning

confidence: 99%

CRISPR/Cas9 Epigenome Editing Potential for Rare Imprinting Diseases: A Review

Syding

Nickl

Kašpárek

et al. 2020

Cells

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Imprinting diseases (IDs) are rare congenital disorders caused by aberrant dosages of imprinted genes. Rare IDs are comprised by a group of several distinct disorders that share a great deal of homology in terms of genetic etiologies and symptoms. Disruption of genetic or epigenetic mechanisms can cause issues with regulating the expression of imprinted genes, thus leading to disease. Genetic mutations affect the imprinted genes, duplications, deletions, and uniparental disomy (UPD) are reoccurring phenomena causing imprinting diseases. Epigenetic alterations on methylation marks in imprinting control centers (ICRs) also alters the expression patterns and the majority of patients with rare IDs carries intact but either silenced or overexpressed imprinted genes. Canonical CRISPR/Cas9 editing relying on double-stranded DNA break repair has little to offer in terms of therapeutics for rare IDs. Instead CRISPR/Cas9 can be used in a more sophisticated way by targeting the epigenome. Catalytically dead Cas9 (dCas9) tethered with effector enzymes such as DNA de- and methyltransferases and histone code editors in addition to systems such as CRISPRa and CRISPRi have been shown to have high epigenome editing efficiency in eukaryotic cells. This new era of CRISPR epigenome editors could arguably be a game-changer for curing and treating rare IDs by refined activation and silencing of disturbed imprinted gene expression. This review describes major CRISPR-based epigenome editors and points out their potential use in research and therapy of rare imprinting diseases.

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Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

Cited by 150 publications

References 72 publications

An improved CRISPR/dCas9 interference tool for neuronal gene suppression

An improved CRISPR/dCas9 interference tool for neuronal gene suppression

Transcriptional enhancers: from prediction to functional assessment on a genome-wide scale

CRISPR/Cas9 Epigenome Editing Potential for Rare Imprinting Diseases: A Review

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