CRISPR/Cas9 system as an innovative genetic engineering tool: Enhancements in sequence specificity and delivery methods

Jo, Young-Il; Suresh, Bharathi; Kim, Hyongbum; Ramakrishna, Suresh

doi:10.1016/j.bbcan.2015.09.003

Cited by 22 publications

(29 citation statements)

References 89 publications

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“…CRISPR spacers recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. As it accurately targets DNA and is a genetically heritable modification, the CRISPR/Cas9 system has been widely used for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation in species throughout life [11, 12]. Furthermore, CRISPR/Cas9 offers great promise for targeting different stages of the viral life cycle, and has the capacity for mediating an effective and sustained genetic therapy against HBV and HIV [25, 26].…”

Section: Discussionmentioning

confidence: 99%

“…Alternative therapeutic strategies of targeting PD-1 or PD-L1 are needed. The clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9-based RNA-guided DNA endonuclease technology has been widely adopted for its affordability, versatility, ease of use, and low cost [11, 12]. CRISPR/Cas9 provides a robust and highly efficient novel genome editing tool, which enables precise manipulation of specific genomic loci, and facilitates elucidation of target gene functions or diseases [13].…”

Section: Introductionmentioning

confidence: 99%

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Targeting programmed cell death ligand 1 by CRISPR/Cas9 in osteosarcoma cells

et al. 2017

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Programmed cell death ligand 1 (PD-L1) is a transmembrane protein that is expressed on tumor cells that suppresses the T cell-mediated immune response. Therapies targeting the PD-L1 pathway promote anti-tumor immunity and have shown promising results in some types of cancers. However, the functional and therapeutic roles of PD-L1 in osteosarcoma remain largely unknown. In this study, we found that PD-L1 protein was expressed in osteosarcoma cell lines and tissue microarray of patient tumors. Tissue microarray immunohistochemistry analysis showed that the overall and five-year survival rates of patients with high levels of PD-L1 expression were significantly shorter than patients with low levels. High levels of PD-L1 expression were also associated with metastasis in osteosarcoma patients. Furthermore, we applied the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system to target PD-L1 gene at the DNA level in osteosarcoma cell lines. We found that the expression of PD-L1 could be efficiently disrupted by CRISPR/Cas9 system and PD-L1 knockdown increased drug sensitivities for doxorubicin and paclitaxel. These results suggest that PD-L1 is an independent prognostic factor in osteosarcoma and that PD-L1 knockout by CRISPR/Cas9 may be a therapeutic approach for the treatment of osteosarcoma.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Targeting programmed cell death ligand 1 by CRISPR/Cas9 in osteosarcoma cells

et al. 2017

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“…47, 48, 49, 51 However, off-target activities depend on several parameters, including the amount of Cas9 protein available, the structure and nature of the sgRNA sequence, the targeted cell type and the cellular state. 77, 78 Off-target activities vary among different cell types and organisms, indicating the need for rigorous studies. Determining the actual frequency of off-target activities is challenging owing to the detection limits of current methodologies.…”

Section: Challenges In Therapeutic Applications Of Molecular Scissorsmentioning

confidence: 99%

“…Overcoming off-target activities by employing multiple strategies and developing highly sensitive detection methods is of paramount importance to the application of Cas9 in genomic medicine. 53, 78, 80 Alternatively, other Cas9 variants with improved specificities might be generated, or other novel classes of endonucleases might be used. 81 …”

Section: Challenges In Therapeutic Applications Of Molecular Scissorsmentioning

confidence: 99%

Genome editing: the road of CRISPR/Cas9 from bench to clinic

Eid

Mahfouz

2016

Exp Mol Med

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Molecular scissors engineered for site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes and could improve our understanding of the molecular underpinnings of disease states and facilitate novel therapeutic applications. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9). The CRISPR/Cas9 system's simplicity, facile engineering and amenability to multiplexing make it the system of choice for many applications. CRISPR/Cas9 has been used to generate disease models to study genetic diseases. Improvements are urgently needed for various aspects of the CRISPR/Cas9 system, including the system's precision, delivery and control over the outcome of the repair process. Here, we discuss the current status of genome engineering and its implications for the future of biological research and gene therapy.

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“…[1][2][3][4] The CRISPR/Cas9 system consists of two critical components: the single-guide RNA (sgRNA) and Cas9 nuclease. [7,8] The breaks are repaired by introducing a sequence to induce the destruction of the sequence or promote the replacement for a healthy target in the presence of donor template DNA. [7,8] The breaks are repaired by introducing a sequence to induce the destruction of the sequence or promote the replacement for a healthy target in the presence of donor template DNA.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9 Delivery Mediated with Hydroxyl‐Rich Nanosystems for Gene Editing in Aorta

Zhang

Gao

et al. 2019

Advanced Science

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A CRISPR/Cas9 system has emerged as a powerful tool for gene editing to treat genetic mutation related diseases. Due to the complete endothelial barrier, effective delivery of the CRISPR/Cas9 system to vasculatures remains a challenge for in vivo gene editing of genetic vascular diseases especially in aorta. Herein, it is reported that CHO‐PGEA (cholesterol (CHO)‐terminated ethanolamine‐aminated poly(glycidyl methacrylate)) with rich hydroxyl groups can deliver a plasmid based pCas9‐sg Fbn 1 system for the knockout of exon 10 in Fbn 1 gene. This is the first report of a polycation‐mediated CRISPR/Cas9 system for gene editing in aorta of adult mice. CHO‐PGEA/pCas9‐sg Fbn 1 nanosystems can effectively contribute to the knockout of exon 10 in Fbn 1 in vascular smooth muscle cells in vitro, which leads to the change of the phosphorylation of Smad2/3 and the increased expression of two downstream signals of Fbn 1: Mmp‐ 2 and Ctgf . For in vivo application, the aortic enrichment of CHO‐PGEA/Cas9‐sg Fbn 1 is achieved by administering a pressor dose of angiotensin II (Ang II). The effects of the pCas9‐sg Fbn 1 system targeting Fbn 1 demonstrate an increase in the expression of Mmp‐ 2 and Ctgf in aorta. Thus, the combination of CHO‐PGEA/pCas9‐sg Fbn 1 nanosystems with Ang II infusion can provide the possibility for in vivo gene editing in aorta.

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CRISPR/Cas9 system as an innovative genetic engineering tool: Enhancements in sequence specificity and delivery methods

Cited by 22 publications

References 89 publications

Targeting programmed cell death ligand 1 by CRISPR/Cas9 in osteosarcoma cells

Targeting programmed cell death ligand 1 by CRISPR/Cas9 in osteosarcoma cells

Genome editing: the road of CRISPR/Cas9 from bench to clinic

CRISPR/Cas9 Delivery Mediated with Hydroxyl‐Rich Nanosystems for Gene Editing in Aorta

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