The Off-Targets of Clustered Regularly Interspaced Short Palindromic Repeats Gene Editing

Vicente, Manuel M.; Chaves-Ferreira, Miguel; Jorge, João M. P.; Proença, João T.; Barreto, Vasco M.

doi:10.3389/fcell.2021.718466

Cited by 15 publications

(14 citation statements)

References 131 publications

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“…Since the death of mice was not related to the loss of function of the target genes, was it due to the defects of RfxCas13d itself? Off-target effects are an ongoing concern for gene editing using any CRISPR-Cas system [ 35 ]. Besides, collateral activity should be considered, although multiple studies have used RfxCas13d in vivo with no reported side effects [ 15 – 17 ].…”

Section: Resultsmentioning

confidence: 99%

The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model

Guo

et al. 2023

Genome Biol

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Background The CRISPR-Cas13 system is an RNA-guided RNA-targeting system and has been widely used in transcriptome engineering with potentially important clinical applications. However, it is still controversial whether Cas13 exhibits collateral activity in mammalian cells. Results Here, we find that knocking down gene expression using RfxCas13d in the adult brain neurons caused death of mice, which may result from the collateral activity of RfxCas13d rather than the loss of target gene function or off-target effects. Mechanistically, we show that RfxCas13d exhibits collateral activity in mammalian cells, which is positively correlated with the abundance of target RNA. The collateral activity of RfxCas13d could cleave 28s rRNA into two fragments, leading to translation attenuation and activation of the ZAKα-JNK/p38-immediate early gene pathway. Conclusions These findings provide new mechanistic insights into the collateral activity of RfxCas13d in mammalian cells and warn that the biosafety of the CRISPR-Cas13 system needs further evaluation before application to clinical treatments.

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Section: Resultsmentioning

confidence: 99%

The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model

Guo

et al. 2023

Genome Biol

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“…It largely depends on the specificity of the introduced corrections (e.g., a proper selection of the target DNA site and the design of single guide RNA in the case of CRISPR/Cas technology), the choice of delivery method (e.g., vector integration ability) and the lack of off-target effects. The general drawbacks of existing genetic engineering techniques have been systemized in a series of comprehensive analyses [ 159 – 162 ] and are beyond the scope of this review. In relation to macrophages, it should be noted that the differentiation of iMphs from iPSCs goes through several stages, includes multiple rounds of cell division, takes at least 2–3 weeks to obtain the first iMph harvest and may last for many months afterwards.…”

Section: Imph-based Cell Therapy: Safety and Other Questions To Explorementioning

confidence: 99%

Macrophages derived from pluripotent stem cells: prospective applications and research gaps

Lyadova

Vasiliev

2022

Cell Biosci

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Induced pluripotent stem cells (iPSCs) represent a valuable cell source able to give rise to different cell types of the body. Among the various pathways of iPSC differentiation, the differentiation into macrophages is a recently developed and rapidly growing technique. Macrophages play a key role in the control of host homeostasis. Their dysfunction underlies many diseases, including hereditary, infectious, oncological, metabolic and other disorders. Targeting macrophage activity and developing macrophage-based cell therapy represent promising tools for the treatment of many pathological conditions. Macrophages generated from human iPSCs (iMphs) provide great opportunities in these areas. The generation of iMphs is based on a step-wise differentiation of iPSCs into mesoderm, hematopoietic progenitors, myeloid monocyte-like cells and macrophages. The technique allows to obtain standardizable populations of human macrophages from any individual, scale up macrophage production and introduce genetic modifications, which gives significant advantages over the standard source of human macrophages, monocyte-derived macrophages. The spectrum of iMph applications is rapidly growing. iMphs have been successfully used to model hereditary diseases and macrophage-pathogen interactions, as well as to test drugs. iMph use for cell therapy is another promising and rapidly developing area of research. The principles and the details of iMph generation have recently been reviewed. This review systemizes current and prospective iMph applications and discusses the problem of iMph safety and other issues that need to be explored before iMphs become clinically applicable.

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“…SpCas9 is the most widely used genome editor, owing to its robust activity in diverse cell types and less stringent PAM requirements. However, SpCas9 tends to display substantial off-target activities compared to other Cas nucleases [ 101 ]. Thus, Cas nucleases from other species, and dCas9- and nCas9-based genome editors that do not induce DSB (i.e., base editors and prime editors) have been extensively investigated for precise genome editing.…”

Section: Small-molecule Enhancers Of Precise Genome Editorsmentioning

confidence: 99%

Small Molecules for Enhancing the Precision and Safety of Genome Editing

2022

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Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome-editing technologies have revolutionized biology, biotechnology, and medicine, and have spurred the development of new therapeutic modalities. However, there remain several barriers to the safe use of CRISPR technologies, such as unintended off-target DNA cleavages. Small molecules are important resources to solve these problems, given their facile delivery and fast action to enable temporal control of the CRISPR systems. Here, we provide a comprehensive overview of small molecules that can precisely modulate CRISPR-associated (Cas) nucleases and guide RNAs (gRNAs). We also discuss the small-molecule control of emerging genome editors (e.g., base editors) and anti-CRISPR proteins. These molecules could be used for the precise investigation of biological systems and the development of safer therapeutic modalities.

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The Off-Targets of Clustered Regularly Interspaced Short Palindromic Repeats Gene Editing

Cited by 15 publications

References 131 publications

The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model

The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model

Macrophages derived from pluripotent stem cells: prospective applications and research gaps

Small Molecules for Enhancing the Precision and Safety of Genome Editing

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