Tools for experimental and computational analyses of off-target editing by programmable nucleases

Bao, Xiaoping; Pan, Yidan; Lee, Ciaran M.; Davis, Timothy; Bao, Gang

doi:10.1038/s41596-020-00431-y

Cited by 66 publications

(59 citation statements)

References 134 publications

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“…Gold-standard methods for assessing off-target impacts include whole genome or ChIP-bisulfite sequencing [ 48 , 49 ]. These methods, although thorough and accurate, are expensive and time-consuming.…”

Section: Resultsmentioning

confidence: 99%

Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System

Smith

Banerjee

Waly

et al. 2021

Cancers

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DNA methylation is a key epigenetic modification implicated in the pathogenesis of numerous human diseases, including cancer development and metastasis. Gene promoter methylation changes are widely associated with transcriptional deregulation and disease progression. The advent of CRISPR-based technologies has provided a powerful toolkit for locus-specific manipulation of the epigenome. Here, we describe a comprehensive global workflow for the design and application of a dCas9-SunTag-based tool for editing the DNA methylation locus in human melanoma cells alongside protocols for downstream techniques used to evaluate subsequent methylation and gene expression changes in methylation-edited cells. Using transient system delivery, we demonstrate both highly efficacious methylation and demethylation of the EBF3 promoter, which is a putative epigenetic driver of melanoma metastasis, achieving up to a 304.00% gain of methylation and 99.99% relative demethylation, respectively. Furthermore, we employ a novel, targeted screening approach to confirm the minimal off-target activity and high on-target specificity of our designed guide RNA within our target locus.

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Section: Resultsmentioning

confidence: 99%

Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System

Smith

Banerjee

Waly

et al. 2021

Cancers

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“…121,127,128 Although prediction tools are not yet perfect, computational algorithms can predict many off-target sites that may be experimentally validated. 129 In silico prediction and biochemical testing with in vitro and/or cellular assays can nominate sets of possible off-target sites to be sequenced to define any experimentally validated off-target sites. 130 A variety of strategies, including modifications to the Cas protein or gRNA or limited temporal exposure to the genome editor (such as with RNP pulse delivery), may reduce the risk of off-target mutagenesis, although there may be trade-offs to consider between ontarget efficiency and specificity.…”

Section: Potential Sources Of Variation In Engraftment Outcomesmentioning

confidence: 99%

Editing outside the body: Ex vivo gene-modification for β-hemoglobinopathy cellular therapy

Rosanwo

Bauer

2021

Molecular Therapy

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“…However, the potential off-target DNA activity needs to be considered especially when developing therapeutic strategies. Methods for detecting off-targets have been recently reviewed [116,117]. Briefly, off-targets of designer nucleases can be predicted using in silico methods, or identified using in vitro or cell-based assays, which can detect DSBs in a genome-wide manner.…”

Section: Off-target Activity Of Genome Editing Systemsmentioning

confidence: 99%

“…In cellular assays are generally believed to be more reliable [118]; however, in most of the cases, surrogate cell lines need to be used because these methods have not been adapted yet to clinically relevant cells, such as HSPCs. As base and prime editors are characterized by little or no DSB generation, the assays off-targets to detect are more limited compared to designer nucleases, but they will probably be developed in the next future [116,117]. Finally, RNAseq is commonly used to detect RNA off-target activity of base editors [100].…”

Section: Off-target Activity Of Genome Editing Systemsmentioning

confidence: 99%

Genome Editing for β-Hemoglobinopathies: Advances and Challenges

Frati

Miccio

2021

JCM

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β-hemoglobinopathies are the most common genetic disorders worldwide and are caused by mutations affecting the production or the structure of adult hemoglobin. Patients affected by these diseases suffer from anemia, impaired oxygen delivery to tissues, and multi-organ damage. In the absence of a compatible donor for allogeneic bone marrow transplantation, the lifelong therapeutic options are symptomatic care, red blood cell transfusions and pharmacological treatments. The last decades of research established lentiviral-mediated gene therapy as an efficacious therapeutic strategy. However, this approach is highly expensive and associated with a variable outcome depending on the effectiveness of the viral vector and the quality of the cell product. In the last years, genome editing emerged as a valuable tool for the development of curative strategies for β-hemoglobinopathies. Moreover, due to the wide range of its applications, genome editing has been extensively used to study regulatory mechanisms underlying globin gene regulation allowing the identification of novel genetic and pharmacological targets. In this work, we review the current advances and challenges of genome editing approaches to β-hemoglobinopathies. Special focus has been directed towards strategies aimed at correcting the defective β-globin gene or at inducing fetal hemoglobin (HbF), which are in an advanced state of clinical development.

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Tools for experimental and computational analyses of off-target editing by programmable nucleases

Cited by 66 publications

References 134 publications

Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System

Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System

Editing outside the body: Ex vivo gene-modification for β-hemoglobinopathy cellular therapy

Genome Editing for β-Hemoglobinopathies: Advances and Challenges

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