Mitigating off-target effects in CRISPR/Cas9-mediated in vivo gene editing

Han, Hua; Pang, Junxiong; Soh, Boon Seng

doi:10.1007/s00109-020-01893-z

Cited by 83 publications

(65 citation statements)

References 205 publications

(275 reference statements)

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“…For more in-depth reviews on this aspect of CRISPR-based therapies see Refs. [230][231][232][233] Regulatory concerns and n-of-1 trials CRISPR-based therapeutics face a host of unique regulatory challenges when compared with other therapeutic methods such as small-molecule drugs or antibodies that typically act at the protein level. There are two layers of information abstraction when moving from DNA to RNA to protein, and often this information may have important implications for therapeutic development.…”

Section: Administrationmentioning

confidence: 99%

Translating CRISPR-Cas Therapeutics: Approaches and Challenges

et al. 2020

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CRISPR-Cas clinical trials have begun, offering a first glimpse at how DNA and RNA targeting could enable therapies for many genetic and epigenetic human diseases. The speedy progress of CRISPR-Cas from discovery and adoption to clinical use is built on decades of traditional gene therapy research and belies the multiple challenges that could derail the successful translation of these new modalities. Here, we review how CRISPR-Cas therapeutics are translated from technological systems to therapeutic modalities, paying particular attention to the therapeutic cascade from cargo to delivery vector, manufacturing, administration, pipelines, safety, and therapeutic target profiles. We also explore potential solutions to some of the obstacles facing successful CRISPR-Cas translation. We hope to illuminate how CRISPR-Cas is brought from the academic bench toward use in the clinic.

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Section: Administrationmentioning

confidence: 99%

Translating CRISPR-Cas Therapeutics: Approaches and Challenges

et al. 2020

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“…To alleviate these kinds of negative side effects, some researchers suggested modifying the length of gRNA aiming to decrease nonspecific INDELs, unwanted insertions, or deletion in the targeted genome [ 88 ]. In a recently completed clinical trial, the length of the gRNA was increased through addition of two guanines to its 5′ end, which resulted in decreased frequency of off-target modifications [ 89 ]. Nonetheless, the modification caused an unwanted decrease in the efficiency of on-target effects.…”

Section: Limitations Of the Crispr/cas9 Systemmentioning

confidence: 99%

CRISPR/Cas9 in Cancer Immunotherapy: Animal Models and Human Clinical Trials

Khalaf

Janowicz

Dyszkiewicz-Konwińska

et al. 2020

Genes

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Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually emerging and evolving mutations of proto-oncogenes and tumor-suppressive genes. CRISPR/Cas9, as a specific gene-editing tool, is used to correct causative mutations with minimal toxicity, but is also employed as an adjuvant to immunotherapy to achieve a more robust immunological response. Some of the most critical limitations of the CRISPR/Cas9 technology include off-target mutations, resulting in nonspecific restrictions of DNA upstream of the Protospacer Adjacent Motifs (PAM), ethical agreements, and the lack of a scientific consensus aiming at risk evaluation. Currently, CRISPR/Cas9 is tested on animal models to enhance genome editing specificity and induce a stronger anti-tumor response. Moreover, ongoing clinical trials use the CRISPR/Cas9 system in immune cells to modify genomes in a target-specific manner. Recently, error-free in vitro systems have been engineered to overcome limitations of this gene-editing system. The aim of the article is to present the knowledge concerning the use of CRISPR Cas9 technique in targeting treatment-resistant cancers. Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology. Demonstrating further, applications and advances of the CRISPR/Cas9 technique are presented in animal models and human clinical trials. Concluding, an overview of the limitations of the gene-editing tool is proffered.

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“…The proper sgRNA design will highly affect the efficacy of the editing system since it determines the activity and specificity of target recognition and, thus, reduces off-target mutations. Many factors contribute to successful sgRNA engineering, including GC content of 40% to 60% and the crRNA length, which controls the level of mismatch tolerance [ 76 ]. Although the general protocol of using the CRISPR/Cas9 system has utilized 20-bp-long crRNA, a 5′ end shortening of the crRNA to 17 or 18 bp has shown a significant reduction of off-target site mutations without the loss of on-target editing activity using WT Cas9 [ 16 ].…”

Section: Principles Of Gene Editing Using Foki–dcas9mentioning

confidence: 99%

“…CRISPR/Cas9 system cellular delivery is one of the main limitations to its wide range of applications [ 82 ]. Lentiviral vectors are commonly used for specific ex-vivo and in-vivo cellular delivery [ 76 ]. However, some disadvantages include the toxicity and immunogenic effects, as well as the limited DNA capacity of about 4.7 kbps that restrains their use [ 76 ].…”

Section: Principles Of Gene Editing Using Foki–dcas9mentioning

confidence: 99%

CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering

Saifaldeen

Al-Ansari

Ramotar

et al. 2020

Cells

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The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR–FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms. Herein, we review the advantages of all published variants of fdCas9 and their current applications in genome engineering.

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Mitigating off-target effects in CRISPR/Cas9-mediated in vivo gene editing

Cited by 83 publications

References 205 publications

Translating CRISPR-Cas Therapeutics: Approaches and Challenges

Translating CRISPR-Cas Therapeutics: Approaches and Challenges

CRISPR/Cas9 in Cancer Immunotherapy: Animal Models and Human Clinical Trials

CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering

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