Detection and quantification of unintended large on-target gene modifications due to CRISPR/Cas9 editing

Cao, Mingming; Bao, Gang

doi:10.1016/j.cobme.2023.100478

Cited by 3 publications

(7 citation statements)

References 52 publications

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“… 30 Currently no single tool can detect all the complex integration outcomes, especially events occurring with very low frequencies. 31 Therefore, we used three assays, FACS, ddPCR, and SMRT-seq with UMI, to comprehensively quantify the molecular events due to gene editing. FACS quantifies the percentage of cells with WAS 2-12 -Pgk-GFP integrated into the genome at on-target and OT sites regardless of the type of integration event.…”

Section: Discussionmentioning

confidence: 99%

Gene editing-based targeted integration for correction of Wiskott-Aldrich syndrome

Pille,

Avila,

Park

et al. 2024

Molecular Therapy - Methods & Clinical Development

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Section: Discussionmentioning

confidence: 99%

Gene editing-based targeted integration for correction of Wiskott-Aldrich syndrome

Pille,

Avila,

Park

et al. 2024

Molecular Therapy - Methods & Clinical Development

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“…Therefore, risk assessment should aim to identify those unintended genetic changes which (for example, in regard to the site, the frequency, its potential gene products or its origin) are unlikely to occur with conventional (non-regulated) methods. The methodology to identify these changes should include WGS by using long read sequencings, also in combination with other methods for gene analysis ( Park et al, 2023 ). Comparison should be performed to the genome of the “wild type” plants that were used as starting point.…”

Section: Discussionmentioning

confidence: 99%

“…It is only afterwards through applying methods, e.g. whole genome sequencing (WGS) and other methods to identify long and short DNA sequence alterations [see, for example, ( Kawall et al, 2020 ; Chu and Agapito-Tenfen, 2022 ; Park et al, 2023 )] that the unintended changes can be detected in the cell. By comparing the “wild type” with the one resulting from NGT applications, the unintended genetic alterations can become detectable and be made comparable to those that are likely to occur with conventional methods.…”

Section: Consequences For a Comprehensive Molecular Characterisation ...mentioning

confidence: 99%

“…By comparing the “wild type” with the one resulting from NGT applications, the unintended genetic alterations can become detectable and be made comparable to those that are likely to occur with conventional methods. As especially large deletions and chromosomal rearrangements are hardly detectable by standard short-range PCR based assays, it is important to combine multiple approaches to assess all types of gene alterations ( Park et al, 2023 ). Park et al (2023) state, no single tool can detect all types of large gene modifications accurately that can be caused by CRISPR/Cas9.…”

Section: Consequences For a Comprehensive Molecular Characterisation ...mentioning

confidence: 99%

“…As especially large deletions and chromosomal rearrangements are hardly detectable by standard short-range PCR based assays, it is important to combine multiple approaches to assess all types of gene alterations ( Park et al, 2023 ). Park et al (2023) state, no single tool can detect all types of large gene modifications accurately that can be caused by CRISPR/Cas9. Therefore, it is important to combine multiple approaches to comprehensively identify and assess the unintended changes throughout the genome [see also ( Mou et al, 2017 ; Hahn and Nekrasov, 2019 ; Yasumoto and Muranaka, 2023 )].…”

Section: Consequences For a Comprehensive Molecular Characterisation ...mentioning

confidence: 99%

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A perspective from the EU: unintended genetic changes in plants caused by NGT—their relevance for a comprehensive molecular characterisation and risk assessment

Koller,

Cieslak

2023

Front. Bioeng. Biotechnol.

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Several regions in the world are currently holding discussions in regard to the regulation of new genomic techniques (NGTs) and their application in agriculture. The European Commission, for instance, is proposing the introduction of specific regulation for NGT plants. Various questions need to be answered including e.g., the extent to which NGT-induced intended and unintended genetic modifications must be subjected to a mandatory risk assessment as part of an approval procedure. This review mostly focuses on findings in regard to unintended genetic changes that can be caused by the application of NGTs. More specifically, the review deals with the application of the nuclease CRISPR/Cas, which is currently the most important tool for developing NGT plants, and its potential to introduce double strand breaks (DSBs) at a targeted DNA sequence. For this purpose, we identified the differences in comparison to non-targeted mutagenesis methods used in conventional breeding. The review concludes that unintended genetic changes caused by NGT processes are relevant to risk assessment. Due to the technical characteristics of NGTs, the sites of the unintended changes, their genomic context and their frequency (in regard to specific sites) mean that the resulting gene combinations (intended or unintended) may be unlikely to occur with conventional methods. This, in turn, implies that the biological effects (phenotypes) can also be different and may cause risks to health and the environment. Therefore, we conclude that the assessment of intended as well as unintended genetic changes should be part of a mandatory comprehensive molecular characterisation and risk assessment of NGT plants that are meant for environmental releases or for market authorisation.

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Guidelines for Performing CRISPR/Cas9 Genome Editing for Gene Validation and Trait Improvement in Crops

Tsakirpaloglou,

Septiningsih,

Thomson

2023

Plants

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With the rapid advances in plant genome editing techniques over the past 10 years, more efficient and powerful crop genome editing applications are now possible. Candidate genes for key traits can be validated using CRISPR/Cas9-based knockouts and through the up- and down-regulation of gene expression. Likewise, new trait improvement approaches can take advantage of targeted editing to improve stress tolerance, disease resistance, and nutritional traits. However, several key steps in the process can prove tricky for researchers who might be new to plant genome editing. Here, we present step-by-step guidelines and best practices for a crop genome editing pipeline that should help to improve the rate of success. Important factors in the process include proper target sequence analysis and single guide RNA (sgRNA) design, sequencing of the target site in the genotypes of interest, performing an in vitro CRISPR/Cas9 ribonucleoprotein (RNP) assay to validate the designed sgRNAs, preparing the transformation constructs, considering a protoplast editing step as further validation, and, finally, stable plant transformation and mutation detection by Sanger and/or next-generation sequencing. With these detailed guidelines, a new user should be able to quickly set up a genome editing pipeline in their crop of interest and start making progress with the different CRISPR/Cas-based editing variants for gene validation and trait improvement purposes.

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Detection and quantification of unintended large on-target gene modifications due to CRISPR/Cas9 editing

Cited by 3 publications

References 52 publications

Gene editing-based targeted integration for correction of Wiskott-Aldrich syndrome

Gene editing-based targeted integration for correction of Wiskott-Aldrich syndrome

A perspective from the EU: unintended genetic changes in plants caused by NGT—their relevance for a comprehensive molecular characterisation and risk assessment

Guidelines for Performing CRISPR/Cas9 Genome Editing for Gene Validation and Trait Improvement in Crops

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