RNA guides genome engineering

Laboratory of Cell and Gene Therapy, Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany.
Nature Biotechnology (Impact Factor: 41.51). 03/2013; 31(3):208-9. DOI: 10.1038/nbt.2527
Source: PubMed
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    • "The desired ultra-specific self-liquidation of transgenic modules can be achieved using CRISPR/Cas9 nucleases, which were proven to be effective and safe in genome editing [18] and are thought of as adequate tools in corrective gene therapy [19]. The self-destruct machinery can be envisaged to consist of: (1) the gene for Cas9 nuclease with genetic elements for its inducible expression; (2) inducible genes to synthesize 'single guide' RNAs (sgRNAs), which direct the endonuclease activity of Cas9 to homologous DNA target sequences [20] [21]; (3) target sequences for the sgRNAs that are scattered throughout the entire transgenic module. Once the expression of the Cas9 gene and the sgRNAs is induced, the whole transgenic unit is shredded into small, genetically-inactive DNA fragments, leaving a double-strand DNA break at the point of initial genomic insertion of the transgenic module. "
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    ABSTRACT: Gene delivery to human somatic cells is a well-established therapeutic strategy to treat a variety of diseases. In addition, gene transfer to human cells is required to generate human induced pluripotent cells and also to eliminate tumorigenic undifferentiated cells in many types of stem-cell derived transplantation material. The expression of transgenes in these medical technologies is often required only in some of the recipient cells and only in specific limited time-windows, with inappropriately located or untimely expressed transgenes presenting a risk of undesired collateral effects. Unfortunately, current gene transfer procedures commonly result in a number of cells in the patient's body containing fragments of transferred genetic material which are either not therapeutically necessary at all, are no longer necessary or are necessary but in some other cells. Such transgenic material in the patient, created as a by-product of the chosen therapeutic procedure, constitutes, in fact, 'genetic litter', that is, persisting potentially-hazardous foreign genetic material which is neither required therapeutically nor explicitly chosen by an informed and free-willing person as an artificial body element. Wider use and more frequent administration of gene and cell therapy in the future are likely to give greater prominence to the issue of misdelivered genetic medicines and of their unwanted remainders accumulating in human bodies. Thus, novel DNA templates, which, on the one hand, are capable of providing transgene expression over broad time-windows, and, on the other hand, do not leave unwanted permanent 'genetic traces', are required. I propose that the problem of 'genetic litter' in patients' bodies can be addressed through the employment of a new type of gene vectors delivering DNA-based transgenic modules with pre-programmed self-destruction. Such vectors could deliver therapeutic DNA cargo and then execute self-liquidation through pre-scheduled activation of co-delivered genome editing tools, such as CRISPR/Cas9 nucleases, specific for the DNA to be eliminated. In this model, all unnecessary transgenic DNA is edited away precisely at a desired time point. Activity of the gene correction apparatus for the specific and effective destruction of transgenic DNA could be turned on by well-timed external signals or could be triggered through intracellular sensors of particular epigenetic signatures. It is expected that the employment of the proposed DNA-based gene vectors equipped with a transgene self-destruct mechanism can extend the safe and ethical application of gene and cell therapy to a broader range of curative and lifestyle-choice medical treatments, e.g., full body prophylactic gene therapy of cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.
    No preview · Article · Aug 2015 · Medical Hypotheses
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    • "Another important concern of the CRISPR/Cas system as an antiviral agent is its specificity [17]. The recognition sequence 5′-N20NGG-3′ of the CRISPR/Cas system is relatively short and mismatches at its 5′-end are tolerable [18], making it prone to induce high off-target effects [17]. In our study, Cas9 induced apoptosis and cellular proliferation inhibition were observed only in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells, suggesting, at least to some extent, that the CRISPR/Cas system was specific enough to distinguish HPV positive cells from HPV negative cells. "
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    ABSTRACT: High-risk human papillomavirus (HR-HPV) has been recognized as a major causative agent for cervical cancer. Upon HPV infection, early genes E6 and E7 play important roles in maintaining malignant phenotype of cervical cancer cells. By using clustered regularly interspaced short palindromic repeats- (CRISPR-) associated protein system (CRISPR/Cas system), a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA) guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells. Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb. Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer.
    Full-text · Article · Jul 2014 · BioMed Research International
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    • "RNA pol III promoters, such as U6 and H1, are commonly used to express these small RNAs. It is believed that mouse U6 promoter transcription starts at the +1 position (23 nt after the TATA box), with G as the preferred initiation nucleotide.23,24,25 However, the exact U6 transcription initiation site has not been rigorously studied. "
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    ABSTRACT: Pol III promoters such as U6 are commonly used to express small RNAs, including small interfering RNA, short hairpin RNA, and guide RNA, for the clustered regularly interspaced short palindromic repeats genome-editing system. However, whether the small RNAs were precisely expressed as desired has not been studied. Here, using deep sequencing to analyze small RNAs, we show that, for mouse U6 promoter, sequences immediately upstream of the putative initiation site, which is often modified to accommodate the restriction enzyme sites that enable easy cloning of small RNAs, are critical for precise transcription initiation. When the promoter is kept unmodified, transcription starts precisely from the first available A or G within the range of positions -1 to +2. In addition, we show that transcription from another commonly used pol III promoter, H1, starts at multiple sites, which results in variability at the 5' end of the transcripts. Thus, inaccuracy of 5' end of small RNA transcripts might be a common problem when using these promoters to express small RNAs based on currently believed concepts. Our study provides general guidelines for minimizing the variability of initiation, thereby enabling more accurate expression of small RNAs.
    Full-text · Article · May 2014 · Molecular Therapy - Nucleic Acids
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