RNA-guided retargeting of Sleeping Beauty transposition in human cells

Kovač, Adrian; Miskey, Csaba; Menzel, Michael; Grueso, Esther; Gogol-Döring, Andreas; Ivics, Zoltán

doi:10.7554/elife.53868

Cited by 50 publications

(42 citation statements)

References 93 publications

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“…The DSB induced by the sequence-specific nucleases at the target sites are fatal in organisms without effective NHEJ or HDR (Xu et al, 2015 ). To overcome this limitation, researchers attempted to connect different transposases to catalytically inactivated dCas9 (site-specific mutations were introduced into the HNH and RuvC domains to abrogate the cleavage activity without affecting the targeted binding effect) to obtain a series of dCas9–transposase fusion proteins [such as dCas9- Himar1 (Chen and Wang, 2019 ), dCas9- piggyBac (Hew et al, 2019 ), and dCas9- Sleeping Beauty (Kovač et al, 2020 )]. These fusions limit the random insertion of transposase, so that transposition occurs only near the point where dCas9 is targeted.…”

Section: Targeting Dsdna By the Type V Familymentioning

confidence: 99%

The Versatile Type V CRISPR Effectors and Their Application Prospects

Tong

Dong

Cui

et al. 2021

Front. Cell Dev. Biol.

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The class II clustered regularly interspaced short palindromic repeats (CRISPR)–Cas systems, characterized by a single effector protein, can be further subdivided into types II, V, and VI. The application of the type II CRISPR effector protein Cas9 as a sequence-specific nuclease in gene editing has revolutionized this field. Similarly, Cas13 as the effector protein of type VI provides a convenient tool for RNA manipulation. Additionally, the type V CRISPR–Cas system is another valuable resource with many subtypes and diverse functions. In this review, we summarize all the subtypes of the type V family that have been identified so far. According to the functions currently displayed by the type V family, we attempt to introduce the functional principle, current application status, and development prospects in biotechnology for all major members.

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Section: Targeting Dsdna By the Type V Familymentioning

confidence: 99%

The Versatile Type V CRISPR Effectors and Their Application Prospects

Tong

Dong

Cui

et al. 2021

Front. Cell Dev. Biol.

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“…Cas9 is a dual RNA-guided DNA endonuclease enzyme that uses base pairing to recognise and cleave target DNA with complementarity to the guide RNA, such as invading bacteriophage DNA or plasmid DNA [ 62 ]. Tethering the transposase toward a target that is overexpressed in the human genome dramatically increases the number of possible attach points and thus induces chances of targeted transposition with a flexible and easy-to-use RNA-guided system [ 63 ]. Pilot studies have indicated that SB is a safe and effective tool to manufacture therapeutic CAR-T cells in cancers [ 64 , 65 , 66 , 67 ].…”

Section: How Are Cars Engineered?mentioning

confidence: 99%

Chimeric Antigen Receptor Design and Efficacy in Ovarian Cancer Treatment

Terlikowska

Dobrzycka

Terlikowski

2021

IJMS

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Our increased understanding of tumour biology gained over the last few years has led to the development of targeted molecular therapies, e.g., vascular endothelial growth factor A (VEGF-A) antagonists, poly[ADP-ribose] polymerase 1 (PARP1) inhibitors in hereditary breast and ovarian cancer syndrome (BRCA1 and BRCA2 mutants), increasing survival and improving the quality of life. However, the majority of ovarian cancer (OC) patients still do not have access to targeted molecular therapies that would be capable of controlling their disease, especially resistant or relapsed. Chimeric antigen receptors (CARs) are recombinant receptor constructs located on T lymphocytes or other immune cells that change its specificity and functions. Therefore, in a search for a successful solid tumour therapy using CARs the specific cell surface antigens identification is crucial. Numerous in vitro and in vivo studies, as well as studies on humans, prove that targeting overexpressed molecules, such as mucin 16 (MUC16), annexin 2 (ANXA2), receptor tyrosine-protein kinase erbB-2 (HER2/neu) causes high tumour cells toxicity and decreased tumour burden. CARs are well tolerated, side effects are minimal and they inhibit disease progression. However, as OC is heterogenic in its nature with high mutation diversity and overexpression of different receptors, there is a need to consider an individual approach to treat this type of cancer. In this publication, we would like to present the history and status of therapies involving the CAR T cells in treatment of OC tumours, suggest potential T cell-intrinsic determinants of response and resistance as well as present extrinsic factors impacting the success of this approach.

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“…Targeted transposition was successful in a bacterial context [ 112 , 113 ], but still remains challenging in eukaryotic cells. By using the CRISPR/Cas9 system with its high targeting efficiency [ 114 ], SB100X transposase fused with catalytically inactive Cas9 (dCas9), and single guide RNAs (sgRNAs) targeting the human Alu retrotransposon, a slight bias towards integration with Alu elements could be accomplished in human cells [ 115 ]. However, the efficiency of the targeted transposition events remained low and further studies are required to bias SB integrations towards defined target regions.…”

Section: New Sleeping Beauty Variants Offeringmentioning

confidence: 99%

Jumping Ahead with Sleeping Beauty: Mechanistic Insights into Cut-and-Paste Transposition

Ochmann

Ivics

2021

Viruses

Self Cite

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Sleeping Beauty (SB) is a transposon system that has been widely used as a genetic engineering tool. Central to the development of any transposon as a research tool is the ability to integrate a foreign piece of DNA into the cellular genome. Driven by the need for efficient transposon-based gene vector systems, extensive studies have largely elucidated the molecular actors and actions taking place during SB transposition. Close transposon relatives and other recombination enzymes, including retroviral integrases, have served as useful models to infer functional information relevant to SB. Recently obtained structural data on the SB transposase enable a direct insight into the workings of this enzyme. These efforts cumulatively allowed the development of novel variants of SB that offer advanced possibilities for genetic engineering due to their hyperactivity, integration deficiency, or targeting capacity. However, many aspects of the process of transposition remain poorly understood and require further investigation. We anticipate that continued investigations into the structure–function relationships of SB transposition will enable the development of new generations of transposition-based vector systems, thereby facilitating the use of SB in preclinical studies and clinical trials.

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RNA-guided retargeting of Sleeping Beauty transposition in human cells

Cited by 50 publications

References 93 publications

The Versatile Type V CRISPR Effectors and Their Application Prospects

The Versatile Type V CRISPR Effectors and Their Application Prospects

Chimeric Antigen Receptor Design and Efficacy in Ovarian Cancer Treatment

Jumping Ahead with Sleeping Beauty: Mechanistic Insights into Cut-and-Paste Transposition

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