Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing

Naeem, Muhammad; Majeed, Saman; Hoque, Mubasher Zahir; Ahmad, Irshad

doi:10.3390/cells9071608

Cited by 328 publications

(211 citation statements)

References 159 publications

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“…Therefore, the GMO Panel considers that the analysis of potential off‐targets would be of very limited value for the risk assessment. In addition, although some biochemical and bioinformatic tools are available to identify potential off‐target mutations (Bae et al., 2014; Tsai et al., 2015; Cameron et al., 2017; Akcakaya et al., 2018; Peng et al., 2018; Naeem et al., 2020), the limited availability and/or completeness of plant genomic sequences and their intraspecies and intravarietal variability would not always allow for a reliable prediction of potential off‐target mutations (Tang et al., 2018; Lee et al., 2019). While an increasing number of publications have investigated off‐target effects for SDN‐based technologies, the GMO Panel noticed that information on the off‐target mechanism and frequency for ODM is quite limited (Modrzejewski et al., 2019).…”

Section: Assessmentmentioning

confidence: 99%

Applicability of the EFSA Opinion on site‐directed nucleases type 3 for the safety assessment of plants developed using site‐directed nucleases type 1 and 2 and oligonucleotide‐directed mutagenesis

Naegeli¹,

Bresson²,

Dalmay³

et al. 2020

EFS2

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The European Commission requested the EFSA Panel on Genetically Modified Organisms (GMO) to assess whether section 4 (hazard identification) and the conclusions of EFSA's Scientific opinion on the risk assessment of plants developed using zinc finger nuclease type 3 technique (ZFN-3) and other site-directed nucleases (SDN) with similar function are valid for plants developed via SDN-1, SDN-2 and oligonucleotide-directed mutagenesis (ODM). In delivering this Opinion, the GMO Panel compared the hazards associated with plants produced via SDN-1, SDN-2 and ODM with those associated with plants obtained via both SDN-3 and conventional breeding. Unlike for SDN-3 methods, the application of SDN-1, SDN-2 and ODM approaches aims to modify genomic sequences in a way which can result in plants not containing any transgene, intragene or cisgene. Consequently, the GMO Panel concludes that those considerations which are specifically related to the presence of a transgene, intragene or cisgene included in section 4 and the conclusions of the Opinion on SDN-3 are not relevant to plants obtained via SDN-1, SDN-2 or ODM as defined in this Opinion. Overall, the GMO Panel did not identify new hazards specifically linked to the genomic modification produced via SDN-1, SDN-2 or ODM as compared with both SDN-3 and conventional breeding. Furthermore, the GMO Panel considers that the existing Guidance for risk assessment of food and feed from genetically modified plants and the Guidance on the environmental risk assessment of genetically modified plants are sufficient but are only partially applicable to plants generated via SDN-1, SDN-2 or ODM. Indeed, those guidance documents' requirements that are linked to the presence of exogenous DNA are not relevant for the risk assessment of plants developed via SDN-1, SDN-2 or ODM approaches if the genome of the final product does not contain exogenous DNA.

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Section: Assessmentmentioning

confidence: 99%

Applicability of the EFSA Opinion on site‐directed nucleases type 3 for the safety assessment of plants developed using site‐directed nucleases type 1 and 2 and oligonucleotide‐directed mutagenesis

Naegeli¹,

Bresson²,

Dalmay³

et al. 2020

EFS2

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“…Genome wide detection techniques, such as Digenome-seq, SITE-seq, and CIRCLE-seq, have been developed to understand the extent of off-target events in an unbiased way [69]. Furthermore, several approaches are now being used to minimize the off-target effect, especially focusing on the CRISPR/Cas9 technology, with optimization of the gRNAs design and the Cas proteins [70]. Point mutations in the sequence of the Cas9 protein have dramatically increased the specificity of the system and generated the so-called high fidelity Cas9 proteins (HiFi Cas9) [71,72].…”

Section: Risks Of Using Gene-editing Techniquesmentioning

confidence: 99%

Gene Augmentation and Editing to Improve TCR Engineered T Cell Therapy against Solid Tumors

et al. 2020

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Recent developments in gene engineering technologies have drastically improved the therapeutic treatment options for cancer patients. The use of effective chimeric antigen receptor T (CAR-T) cells and recombinant T cell receptor engineered T (rTCR-T) cells has entered the clinic for treatment of hematological malignancies with promising results. However, further fine-tuning, to improve functionality and safety, is necessary to apply these strategies for the treatment of solid tumors. The immunosuppressive microenvironment, the surrounding stroma, and the tumor heterogeneity often results in poor T cell reactivity, functionality, and a diminished infiltration rates, hampering the efficacy of the treatment. The focus of this review is on recent advances in rTCR-T cell therapy, to improve both functionality and safety, for potential treatment of solid tumors and provides an overview of ongoing clinical trials. Besides selection of the appropriate tumor associated antigen, efficient delivery of an optimized recombinant TCR transgene into the T cells, in combination with gene editing techniques eliminating the endogenous TCR expression and disrupting specific inhibitory pathways could improve adoptively transferred T cells. Armoring the rTCR-T cells with specific cytokines and/or chemokines and their receptors, or targeting the tumor stroma, can increase the infiltration rate of the immune cells within the solid tumors. On the other hand, clinical “off-tumor/on-target” toxicities are still a major potential risk and can lead to severe adverse events. Incorporation of safety switches in rTCR-T cells can guarantee additional safety. Recent clinical trials provide encouraging data and emphasize the relevance of gene therapy and gene editing tools for potential treatment of solid tumors.

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“…Altering SpCas9 to increase specificity, change PAMs, or improve efficiency has been of high interest in the genome editing world (Naeem et al, 2020). For example, a version of SpCas9, xCas9, was developed to recognize a wider array of PAMs increasing the sites that could be targeted with the protein.…”

Section: Cas Variationsmentioning

confidence: 99%

“…There are many tools that have been developed by industry and academic groups to computationally predict where in a genome a targeting component, such as a sgRNA or TALEN, will bind to (Bradford & Perrin, 2019, Bogdanove & Booher, 2016. The variety of target prediction tools ranging from scoringbased to alignment-based models that are current developed for CRISPR/Cas9 was recently summarized by Naeem et al (2020). Use of these computational tools is both crucial and common practice when designing components to reduce the possibility of off-target edits and to identify genomic locations that will need to be checked for edits if whole genome sequencing is not feasible.…”

Section: Target Selectionmentioning

confidence: 99%

“…Furthermore, there is a myriad of other techniques that can be used to test for genomewide DSBs caused by a genome editing tool (Naeem et al, 2020). One possibility for testing efficiency of genome editing tools in any organism's genome is to use in vitro methods to test out potential off-targets in a cell-free way (Cameron et al, 2017, Tsai et al, 2017.…”

Section: Preliminary Testing For Edits and Off-targetsmentioning

confidence: 99%

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Current Capabilities, Precision, and Risks of Genome Editing Techniques in Agricultural Systems

Carter¹

2020

Preprint

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We are in a new chapter of crop and livestock improvement with the emergence of genome editing. This latest generation of molecular tools can be used to make targeted changes in a genome including insertions, deletions, and mutations. With new advances comes new risks for unintended changes and impacts, thus the need for appropriate risk assessment for product development and to inform regulatory measures. Though CRISPR/Cas has arisen as the predominant technology, there are multiple types of genome editing tools each with pros and cons depending on the organism and desired outcome. Furthermore, each editing tool differs in specificity as they may edit non-intended sites, referred to as off-target edits. The consensus of the agricultural editing community is to avoid off-target editing through design and detection, instead of determining whether off-target editing in each case is detrimental. The design of a targeting component, the tool chosen, and the identification of the edit(s) made are the critical factors in avoiding off-target edits and confirming intended edits in final products that are released commercially. The limited amount of head-to-head comparisons of genome editing tools in diverse crops and livestock make it difficult to develop broad conclusions and best practices, which is further compounded by the diversity of techniques, targets, and processes. Developers and breeders should consult the literature and test as needed to determine which editing technology will be the most effective for their purposes, especially as more tools with altered efficiency and specificity become available. Yet, the lack of off-target edits in studies that employed careful design of targeting components followed by wide testing for on- and off-target edits bodes well for the use of genome editing with proper precautions of target selection and screening.

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Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing

Cited by 328 publications

References 159 publications

Applicability of the EFSA Opinion on site‐directed nucleases type 3 for the safety assessment of plants developed using site‐directed nucleases type 1 and 2 and oligonucleotide‐directed mutagenesis

Applicability of the EFSA Opinion on site‐directed nucleases type 3 for the safety assessment of plants developed using site‐directed nucleases type 1 and 2 and oligonucleotide‐directed mutagenesis

Gene Augmentation and Editing to Improve TCR Engineered T Cell Therapy against Solid Tumors

Current Capabilities, Precision, and Risks of Genome Editing Techniques in Agricultural Systems

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