In vivo epigenome editing and transcriptional modulation using CRISPR technology

Lau, Cia-Hin; Suh, Yousin

doi:10.1007/s11248-018-0096-8

Cited by 30 publications

(17 citation statements)

References 117 publications

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“…Transcriptional modulation offers several advantages over genome editing, such as restoring normal gene expression without inducing DNA damage and off-target mutagenesis. With the exception of gene knockout, 39 no study yet has reported the use of an all-in-one AAV system for the delivery of CRISPR fusion protein and its sgRNA into the adult mouse brain 40 . To our knowledge, the existing strategies to enable transcriptional modulation in vivo were based on the use of a dual-vector AAV system, 41 a split dCas9 AAV system,42, 43 or Cre-inducible dCas9-expressing mice 44, 45, 46.…”

Section: Discussionmentioning

confidence: 99%

Targeted Transgene Activation in the Brain Tissue by Systemic Delivery of Engineered AAV1 Expressing CRISPRa

Lau

et al. 2019

Molecular Therapy - Nucleic Acids

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Targeted transcriptional modulation in the central nervous system (CNS) can be achieved by adeno-associated virus (AAV) delivery of CRISPR activation (CRISPRa) and interference (CRISPRi) transgenes. To enable AAV packaging, we constructed minimal CRISPRa and CRISPRi transgenes by fusing catalytically inactive Staphylococcus aureus Cas9 (dSaCas9) to the transcriptional activator (VP64 and VP160) and repressor (KRAB and SID4X) domains along with truncated regulatory elements. We then evaluated the performance of these constructs in two reporter assays (bioluminescent and fluorescent), five endogenous genes ( Camk2a , Mycn , Nrf2 , Keap1 , and PDGFRA ), and two cell lines (neuro-2a [N2a] and U87) by targeting the promoter and/or enhancer regions. To enable systemic delivery of AAVs to the CNS, we have also generated an AAV1-PHP.B by inserting a 7-mer PHP.B peptide on AAV1 capsid. We showed that AAV1-PHP.B can efficiently cross the blood-brain barrier (BBB) and be taken up by the brain tissue upon lateral tail vein injection in mice. Importantly, a single-dose intravenous administration of AAV1-PHP.B expressing CRISPRa was shown to achieve targeted transgene activation in the mouse brain. This proof-of-concept study will contribute to the development of a non-invasive, specific and potent AAV-CRISPR system for correcting transcriptional misregulation in broad brain areas and multiple neuroanatomical structures.

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Section: Discussionmentioning

confidence: 99%

Targeted Transgene Activation in the Brain Tissue by Systemic Delivery of Engineered AAV1 Expressing CRISPRa

Lau

et al. 2019

Molecular Therapy - Nucleic Acids

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“…Additionally, catalytically inactive Cas9 has been fused to various epigenetic effectors such as the catalytic core of the human acetyltransferase p300 which catalyzes acetylation of histone H3 lysine 27 ( Hilton et al, 2015 ), histone demethylase ( Kearns et al, 2015 ), histone deacetylase (HDAC) ( Kwon et al, 2017 ) and many others (reviewed in Lau and Suh, 2018 ).…”

Section: Transcriptional Modulation and Epigenome Editingmentioning

confidence: 99%

Expanding the CRISPR Toolbox in Zebrafish for Studying Development and Disease

Liu

Petree

Requena

et al. 2019

Front. Cell Dev. Biol.

118

100

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The study of model organisms has revolutionized our understanding of the mechanisms underlying normal development, adult homeostasis, and human disease. Much of what we know about gene function in model organisms (and its application to humans) has come from gene knockouts: the ability to show analogous phenotypes upon gene inactivation in animal models. The zebrafish (Danio rerio) has become a popular model organism for many reasons, including the fact that it is amenable to various forms of genetic manipulation. The RNA-guided CRISPR/Cas9-mediated targeted mutagenesis approaches have provided powerful tools to manipulate the genome toward developing new disease models and understanding the pathophysiology of human diseases. CRISPR-based approaches are being used for the generation of both knockout and knock-in alleles, and also for applications including transcriptional modulation, epigenome editing, live imaging of the genome, and lineage tracing. Currently, substantial effort is being made to improve the specificity of Cas9, and to expand the target coverage of the Cas9 enzymes. Novel types of naturally occurring CRISPR systems [Cas12a (Cpf1); engineered variants of Cas9, such as xCas9 and SpCas9-NG], are being studied and applied to genome editing. Since the majority of pathogenic mutations are single point mutations, development of base editors to convert C:G to T:A or A:T to G:C has further strengthened the CRISPR toolbox. In this review, we provide an overview of the increasing number of novel CRISPR-based tools and approaches, including lineage tracing and base editing.

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“…DNA methylation is initiated by DNA methyltransferase (DNMT), so controlling DNMT activity should in turn control DNA methylation. By fusing dCas9 to a catalytic domain of DNMT, such as DNMT3 or MQ3, CRISPR/Cas systems have successfully targeted methylation to specific sites in both plants and animals ( Liu et al, 2016 ; Vojta et al, 2016 ; Lau and Suh, 2018 ). By fusing dCas to a demethylation enzyme, such as a ten-eleven translocation (TET) family member, the CRISPR/dCas system may also be used to remove methyl groups from specific DNA sequences, initiating increased expression of the target gene.…”

Section: Crispr/cas Precision Genome Editingmentioning

confidence: 99%

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

Brant

Budak

et al. 2021

J. Zhejiang Univ. Sci. B

110

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Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010s, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has rapidly been developed into a robust, multifunctional genome editing tool with many uses. Following the discovery of the initial CRISPR/Cas-based system, the technology has been advanced to facilitate a multitude of different functions. These include development as a base editor, prime editor, epigenetic editor, and CRISPR interference (CRISPRi) and CRISPR activator (CRISPRa) gene regulators. It can also be used for chromatin and RNA targeting and imaging. Its applications have proved revolutionary across numerous biological fields, especially in biomedical and agricultural improvement. As a diagnostic tool, CRISPR has been developed to aid the detection and screening of both human and plant diseases, and has even been applied during the current coronavirus disease 2019 (COVID-19) pandemic. CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases, including cancers, and has aided drug development. In terms of agricultural breeding, precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins, starch, oil, and other functional components for crop improvement. Adding to this, CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators. Looking to the future, increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology. This review provides an in-depth overview of current CRISPR development, including the advantages and disadvantages of the technology, recent applications, and future considerations.

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In vivo epigenome editing and transcriptional modulation using CRISPR technology

Cited by 30 publications

References 117 publications

Targeted Transgene Activation in the Brain Tissue by Systemic Delivery of Engineered AAV1 Expressing CRISPRa

Targeted Transgene Activation in the Brain Tissue by Systemic Delivery of Engineered AAV1 Expressing CRISPRa

Expanding the CRISPR Toolbox in Zebrafish for Studying Development and Disease

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

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