Efficient genome editing in primary cells and <i>in vivo</i> using viral-derived “Nanoblades” loaded with Cas9/sgRNA ribonucleoproteins

Mangeot, Philippe; Rissons, Valerie; Fusil, Floriane; Marnef, Aline; Laurent, Emilie; Blin, Juliana; Mournetas, Virginie; Massouridès, Emmanuelle; Sohier, Thibault J.M.; Corbin, Antoine; Aubé, Fabien; Pinset, Christian; Schaeffer, Laurent; Legube, Gaëlle; Cosset, François‐Loïc; Verhoeyen, Els; Ohlmann, Théophile; Ricci, Emiliano P.

doi:10.1101/202010

Cited by 7 publications

(8 citation statements)

References 32 publications

(30 reference statements)

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“…Toward this end, many delivery modalities have been developed and optimized to transduce clinically relevant cell types in vivo (Long et al, 2016;Goldstein et al, 2019;Mangeot et al, 2019;Gillmore et al, 2021). These platforms are now being used to package and deliver CRISPR-based editing tools in vivo, which has shown initial success in the first human clinical trials.…”

Section: Methods To Identify Unintended Editing Events In Vivomentioning

confidence: 99%

CRISPR nuclease off-target activity and mitigation strategies

Wienert¹,

Cromer

2022

Front. Genome Ed.

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The discovery of CRISPR has allowed site-specific genomic modification to become a reality and this technology is now being applied in a number of human clinical trials. While this technology has demonstrated impressive efficacy in the clinic to date, there remains the potential for unintended on- and off-target effects of CRISPR nuclease activity. A variety of in silico-based prediction tools and empirically derived experimental methods have been developed to identify the most common unintended effect—small insertions and deletions at genomic sites with homology to the guide RNA. However, large-scale aberrations have recently been reported such as translocations, inversions, deletions, and even chromothripsis. These are more difficult to detect using current workflows indicating a major unmet need in the field. In this review we summarize potential sequencing-based solutions that may be able to detect these large-scale effects even at low frequencies of occurrence. In addition, many of the current clinical trials using CRISPR involve ex vivo isolation of a patient’s own stem cells, modification, and re-transplantation. However, there is growing interest in direct, in vivo delivery of genome editing tools. While this strategy has the potential to address disease in cell types that are not amenable to ex vivo manipulation, in vivo editing has only one desired outcome—on-target editing in the cell type of interest. CRISPR activity in unintended cell types (both on- and off-target) is therefore a major safety as well as ethical concern in tissues that could enable germline transmission. In this review, we have summarized the strengths and weaknesses of current editing and delivery tools and potential improvements to off-target and off-tissue CRISPR activity detection. We have also outlined potential mitigation strategies that will ensure that the safety of CRISPR keeps pace with efficacy, a necessary requirement if this technology is to realize its full translational potential.

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Section: Methods To Identify Unintended Editing Events In Vivomentioning

confidence: 99%

CRISPR nuclease off-target activity and mitigation strategies

Wienert¹,

Cromer

2022

Front. Genome Ed.

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“…Methods to deliver gene editing tools as RNPs offer advantages such as increased safety and packaging capacity. Several promising vehicles for protein delivery have emerged including LPs (Cai et al, 2014), nanoblades (Mangeot et al, 2019) and most recently, virus-like particles (VLPs) (Banskota et al, 2022). However, protein delivery of editing tools to the target tissue has been shown to be a limitation that significantly impacts the in vivo editing efficiency of these techniques.…”

Section: Applications Of This Methodsmentioning

confidence: 99%

Simple Autofluorescence-Restrictive Sorting of eGFP+ RPE Cells Allows Reliable Assessment of Targeted Retinal Gene Therapy

Alsing

Lindholm

Haldrup

et al. 2022

Front. Drug. Deliv.

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Gene therapy is a promising therapeutic modality for ocular diseases arising in and affecting the retina and choroid. In this context, delivering gene therapy to the multifunctional retinal pigment epithelium (RPE) cells situated between the retina and choroid is desired. Efficacy assessment of any gene therapy strategy, whether it is gene augmentation, inhibition, or editing is initially tested in vitro in cell models, where delivery is simple and efficient. However, efficacy assessment in vivo in animal models is far more complex and several factors can influence the result significantly. Here we report a simple fluorescence activated cell sorting (FACS)-based enrichment method for direct assessment of efficacy and potential off-target effects of gene therapy co-delivered with an eGFP reporter to murine RPE cells using subretinal administration. Isolation of true eGFP+ RPE cells by FACS is notoriously difficult due to their intrinsic autofluorescence resulting in decreased sensitivity and false positives. Combining retinal dissection and harvest of RPE cells with a FACS-gating strategy utilizing the GFP filter and a neighboring filter, to separate the eGFP signal from autofluorescence, allows a significant enrichment of gene therapy-targeted eGFP+ RPE cells. In our hands the method may provide quantitative and qualitative advances in terms of up to 7-fold enrichment of true eGFP+ RPE cells compared to a standard protocol. The isolated cells can subsequently be utilized for reliable assessment of changes in DNA, RNA, or protein. This method allows proof-of-principle analysis of early gene therapy development and investigation of new delivery strategies or therapeutic approaches targeting RPE cells in vivo.

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“…Whether in vitro or in vivo, extracellular vesicles (EVs) have been widely used to efficiently deliver genes or drugs (Choi et al, 2016;Montagna et al, 2018;Campbell et al, 2019;Mangeot et al, 2019;Gee et al, 2020). As natural cell-derived membrane vesicles, EVs serve the function of cell-to-cell communication with outstanding biocompatibility and immune-privileged characteristics.…”

Section: Extracellular Vesiclesmentioning

confidence: 99%

Strategies for High-Efficiency Mutation Using the CRISPR/Cas System

Feng

Wang

et al. 2022

Front. Cell Dev. Biol.

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Clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems have revolutionized traditional gene-editing tools and are a significant tool for ameliorating gene defects. Characterized by high target specificity, extraordinary efficiency, and cost-effectiveness, CRISPR/Cas systems have displayed tremendous potential for genetic manipulation in almost any organism and cell type. Despite their numerous advantages, however, CRISPR/Cas systems have some inherent limitations, such as off-target effects, unsatisfactory efficiency of delivery, and unwanted adverse effects, thereby resulting in a desire to explore approaches to address these issues. Strategies for improving the efficiency of CRISPR/Cas-induced mutations, such as reducing off-target effects, improving the design and modification of sgRNA, optimizing the editing time and the temperature, choice of delivery system, and enrichment of sgRNA, are comprehensively described in this review. Additionally, several newly emerging approaches, including the use of Cas variants, anti-CRISPR proteins, and mutant enrichment, are discussed in detail. Furthermore, the authors provide a deep analysis of the current challenges in the utilization of CRISPR/Cas systems and the future applications of CRISPR/Cas systems in various scenarios. This review not only serves as a reference for improving the maturity of CRISPR/Cas systems but also supplies practical guidance for expanding the applicability of this technology.

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Efficient genome editing in primary cells and in vivo using viral-derived “Nanoblades” loaded with Cas9/sgRNA ribonucleoproteins

Cited by 7 publications

References 32 publications

CRISPR nuclease off-target activity and mitigation strategies

CRISPR nuclease off-target activity and mitigation strategies

Simple Autofluorescence-Restrictive Sorting of eGFP+ RPE Cells Allows Reliable Assessment of Targeted Retinal Gene Therapy

Strategies for High-Efficiency Mutation Using the CRISPR/Cas System

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