An enhanced CRISPR repressor for targeted mammalian gene regulation

Yeo, Nan Cher; Chavez, Alejandro; Lance-Byrne, Alissa; Chan, Yingleong; Menn, David J.; Milanova, Denitsa; Kuo, Chih Chung; Guo, Xiaoge; Sharma, Sumana; Tung, Angela; Cecchi, Ryan J; Tuttle, Marcelle; Pradhan, Swechchha; Lim, Elaine T.; Davidsohn, Noah; Ebrahimkhani, Mo R.; Collins, James J.; Lewis, Nathan E.; Kiani, Samira; Church, George M.

doi:10.1038/s41592-018-0048-5

Cited by 428 publications

(354 citation statements)

References 54 publications

(45 reference statements)

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“…Double Cas9 excision of DNA sequences that flank lncRNAs are more likely to inactivate lncRNA gene function and has been used to delete up to hundreds of human lncRNAs, revealing the function of previously uncharacterized lncRNA loci [26,88,89]. Engineered Cas9 variants, in particular dCas9 fused to transcriptional activators or repressors, are highly effective for modulating the expression of lncRNAs without alterations to the underlying genomic DNA sequence [90][91][92][93][94][95][96]. The CRISPRi system (Fig 2G), in which dCas9 is fused with the KRAB repressor domain (dCas9-KRAB), silences transcription through steric blockade of RNA polymerase elongation and local deposition of H3K9me3, which is a characteristic heterochromatin mark [90,91,97].…”

Section: Engineered Crispr Methods Of Decreasing Lncrna Expressionmentioning

confidence: 99%

Modulating the expression of long non‐coding RNA s for functional studies

Liu

Lim

2018

EMBO Reports

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Long non‐coding RNAs (lncRNAs) have emerged as important regulators of cell biology. The mechanisms by which lncRNAs function are likely numerous, and most are poorly understood. Currently, the mechanisms of functional lncRNAs include those that directly involve the lncRNA transcript, the process of their own transcription and splicing, and even underlying transcriptional regulatory elements within the genomic DNA that encodes the lncRNA. As our understanding of lncRNA biology evolves, so have the methods that are utilized to elucidate their functions. In this review, we survey a collection of different methods used to modulate lncRNA expression levels for the assessment of biological function. From RNA‐targeted strategies, genetic deletions, to engineered gene regulatory systems, the advantages and caveats of each method will be discussed. Ultimately, the selection of tools will be guided by which potential lncRNA mechanisms are being investigated, and no single method alone will likely be sufficient to reveal the function of any particular lncRNA.

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Section: Engineered Crispr Methods Of Decreasing Lncrna Expressionmentioning

confidence: 99%

Modulating the expression of long non‐coding RNA s for functional studies

Liu

Lim

2018

EMBO Reports

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“…dCas9 is then fused to different effector molecules that enable a variety of different applications including transcriptional control (e.g. VPR or KRAB) (Gilbert et al , ; Chavez et al , ; Yeo et al , ), epigenome modification (e.g. p300 HAT domain) (Hilton et al , ), fluorescent tracking (e.g.…”

Section: Future Directionsmentioning

confidence: 99%

Therapeutic gene editing in haematological disorders with CRISPR/Cas9

2019

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Summary The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR‐associated (Cas)9 platform offers an efficient way of making precise genetic changes to the human genome. This can be employed for disruption, addition and correction of genes, thereby enabling a new class of genetic therapies that can be applied to haematological disorders. Here we review recent technological advances in the CRISPR/Cas9 methodology and applications in haematology for curing monogenic genetic disorders and for engineering novel chimeric antigen receptor (CAR) T cells to treat haematological malignancies. Furthermore, we discuss current challenges for full clinical implementation of CRISPR/Cas9, and reflect on future trajectories of the technology.

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“…Recently, major advances have been made in the modulation of endogenous gene expression by repurposing the CRISPR/Cas9 system [17][18][19][20][21][22][23][24][25][26][27][28][29] . By coupling the deactivated Cas9 (dCas9) to various activator (e.g.…”

Section: Introductionmentioning

confidence: 99%

Interrogation of Enhancer Function by Enhanced CRISPR Epigenetic Editing

Liu

Cao

et al. 2019

Preprint

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Tissue-specific gene expression requires coordinated control of gene-proximal and -distal cisregulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging. Here we describe enhanced CRISPR/dCas9-based epigenetic editing systems, enCRISPRa and enCRISPRi, for multiplexed analysis of enhancer function in situ and in vivo. Using dual effectors capable of re-writing enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, the new systems display more robust perturbation of enhancer activity and gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Multiplexed perturbations of lineage-specific enhancers using an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted essentiality of developmental enhancers during hematopoietic lineage specification. Hence, enhanced CRSIPR epigenetic editing provides opportunities for interrogating enhancer function in native biological contexts. 16 weeks Controls Enhancers Promoters sgRNAs targeting: Controls Enhancers Promoters sgRNAs targeting: Controls Enhancers Promoters sgRNAs targeting: Controls Enhancers Promoters sgRNAs targeting: Controls Enhancers Promoters sgRNAs targeting: Controls Enhancers Promoters sgRNAs targeting: Controls Enhancers Promoters sgRNAs targeting:

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An enhanced CRISPR repressor for targeted mammalian gene regulation

Cited by 428 publications

References 54 publications

Modulating the expression of long non‐coding RNA s for functional studies

Modulating the expression of long non‐coding RNA s for functional studies

Therapeutic gene editing in haematological disorders with CRISPR/Cas9

Interrogation of Enhancer Function by Enhanced CRISPR Epigenetic Editing

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