TraFo-CRISPR: Enhanced Genome Engineering by Transient Foamy Virus Vector-Mediated Delivery of CRISPR/Cas9 Components

Lindel, Fabian; Dodt, Carolin; Weidner, Niklas; Noll, Monique; Bergemann, Fabian; Behrendt, Rayk; Fischer, Sarah; Dietrich, Josephine; Cartellieri, Marc; Hamann, Martin V.; Lindemann, Dirk

doi:10.1016/j.omtn.2019.10.006

Cited by 15 publications

(26 citation statements)

References 88 publications

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“…[73][74][75][76][77] The possible reasons are the heterogeneity of T cells and tumor cells and their complex interactions in the tumor microenvironment. [78][79][80][81] Immunogenomics is a relatively new field of cancer research. The detection and analysis of whole-genome sequencing (WGS), whole-exome sequencing (WES), and RNA sequencing (RNA-Seq) on T cells and tumor cells by NGS technology can obtain genome maps of tumors and immune cells, which can help to customize treatment schemes for specific characteristics of tumors and increase the possibility of success.…”

Section: Next-generation Sequencing and Immunotherapy For Tumorsmentioning

confidence: 99%

Power and Promise of Next-Generation Sequencing in Liquid Biopsies and Cancer Control

Liu

et al. 2020

Cancer Control

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Traditional methods of cancer treatment are usually based on the morphological and histological diagnosis of tumors, and they are not optimized according to the specific situation. Precision medicine adjusts the existing treatment regimen based on the patient’s genomic information to make it most suitable for patients. Detection of genetic mutations in tumors is the basis of precise cancer medicine. Through the analysis of genetic mutations in patients with cancer, we can tailor the treatment plan for each patient with cancer to maximize the curative effect, minimize damage to healthy tissues, and optimize resources. In recent years, next-generation sequencing technology has developed rapidly and has become the core technology of precise targeted therapy and immunotherapy for cancer. From early cancer screening to treatment guidance for patients with advanced cancer, liquid biopsy is increasingly used in cancer management. This is as a result of the development of better noninvasive, repeatable, sensitive, and accurate tools used in early screening, diagnosis, evaluation, and monitoring of patients. Cell-free DNA, which is a new noninvasive molecular pathological detection method, often carries tumor-specific gene changes. It plays an important role in optimizing treatment and evaluating the efficacy of different treatment options in clinical trials, and it has broad clinical applications.

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Section: Next-generation Sequencing and Immunotherapy For Tumorsmentioning

confidence: 99%

Power and Promise of Next-Generation Sequencing in Liquid Biopsies and Cancer Control

Liu

et al. 2020

Cancer Control

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“…Indeed, the titers we obtained with these vector batches remained high, and they are in the best design configuration for efficient gene editing. Other groups used similar bacteriophage systems to create particles that can transfer RNA into target cells using a slightly different design compared with ours [ 15 , 18 , 19 ]. Briefly, Knopp et al replaced the nucleocapsid by a genetically fused MS2 heterodimer to generate non-integrating gammaretroviral murine leukemia virus-based CRISPR/Cas9 all-in-one particles.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-mediated gene knockout and interallelic gene conversion in human induced pluripotent stem cells using non-integrative bacteriophage-chimeric retrovirus-like particles

et al. 2022

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Background The application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications. Results We found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion. Conclusions Our results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.

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“…Taking advantage of the foamy viruses to efficiently package non-viral cellular RNAs [ 86 , 87 ], Lindel et al successfully used foamy viral capsid to package and deliver SpCas9 mRNA [ 49 ]. They observed > 80% genome editing activity and improved specificity compared with viral delivery.…”

Section: Using Vlps As Safe Gene Editing Delivery Vehiclesmentioning

confidence: 99%

“…So far, VLP-mediated Cas9 mRNA delivery to mammalian cells was more successful than sgRNA delivery. Several studies have found that sgRNA packaged alone could not be functionally delivered [ 47 , 49 , 55 ]. Single guide RNA is very unstable in cells unless complexed with Cas9 protein [ 88 ].…”

Section: Using Vlps As Safe Gene Editing Delivery Vehiclesmentioning

confidence: 99%

“…Single guide RNA is very unstable in cells unless complexed with Cas9 protein [ 88 ]. Due to the inability to package sgRNA by viral capsids, sgRNA has to be delivered via traditional methods, such as plasmid DNA transfection and integration-defective lentiviral vectors [ 45 , 46 , 47 , 48 , 49 ].…”

Section: Using Vlps As Safe Gene Editing Delivery Vehiclesmentioning

confidence: 99%

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Virus-Like Particle Mediated CRISPR/Cas9 Delivery for Efficient and Safe Genome Editing

Lyu

Wang

2020

Life

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The discovery of designer nucleases has made genome editing much more efficient than before. The designer nucleases have been widely used for mechanistic studies, animal model generation and gene therapy development. However, potential off-targets and host immune responses are issues still need to be addressed for in vivo uses, especially clinical applications. Short term expression of the designer nucleases is necessary to reduce both risks. Currently, various delivery methods are being developed for transient expression of designer nucleases including Zinc Finger Nuclease (ZNF), Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas). Recently, virus-like particles are being used for gene editing. In this review, we will talk through commonly used genome editing nucleases, discuss gene editing delivery tools and review the latest literature using virus-like particles to deliver gene editing effectors.

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TraFo-CRISPR: Enhanced Genome Engineering by Transient Foamy Virus Vector-Mediated Delivery of CRISPR/Cas9 Components

Cited by 15 publications

References 88 publications

Power and Promise of Next-Generation Sequencing in Liquid Biopsies and Cancer Control

Power and Promise of Next-Generation Sequencing in Liquid Biopsies and Cancer Control

CRISPR/Cas9-mediated gene knockout and interallelic gene conversion in human induced pluripotent stem cells using non-integrative bacteriophage-chimeric retrovirus-like particles

Virus-Like Particle Mediated CRISPR/Cas9 Delivery for Efficient and Safe Genome Editing

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