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Delivery of CRISPR/Cas9 system by AAV as vectors for gene therapy.
The adeno-associated virus (AAV) is a defective single-stranded DNA virus with the simplest structure reported to date. It constitutes a capsid protein and single-stranded DNA. With its high transduction efficiency, low immunogenicity, and tissue specificity, it is the most widely used and promising gene therapy vector. The clustered regularly interspaced short palindromic sequence (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system is an emerging technology that utilizes cas9 nuclease to specifically recognize and cleave target genes under the guidance of small guide RNA and realizes gene editing through homologous directional repair and non-homologous recombination repair. In recent years, an increasing number of animal experiments and clinical studies have revealed the great potential of AAV as a vector to deliver the CRISPR/cas9 system for treating genetic diseases and viral infections. However, the immunogenicity, toxicity, low transmission efficiency in brain and ear tissues, packaging size limitations of AAV, and immunogenicity and off-target effects of Cas9 protein pose several clinical challenges. This research reviews the role, challenges, and countermeasures of the AAV-CRISPR/cas9 system in gene therapy.
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Comment on "Why are NGOs sceptical of genome editing?".
Comment on “Why are NGOs sceptical of genome editing?” by Helliwell et al.[Image: see text]
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Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development.
A standard approach in the identification of transcriptional enhancers is the use of transgenic animals carrying DNA elements joined to reporter genes inserted randomly in the genome. We examined elements near Tbx5, a gene required for forelimb development in humans and other vertebrates. Previous transgenic studies reported a mammalian Tbx5 forelimb enhancer located in intron 2 containing a putative retinoic acid response element and a zebrafish tbx5a forelimb (pectoral fin) enhancer located downstream that is conserved from fish to mammals. We used CRISPR/Cas9 gene editing to knockout the endogenous elements and unexpectedly found that deletion of the intron 2 and downstream elements, either singly or together in double knockouts, resulted in no effect on forelimb development. Our findings show that reporter transgenes may not identify endogenous enhancers and that in vivo genetic loss-of-function studies are required, such as CRISPR/Cas9, which is similar in effort to production of animals carrying reporter transgenes.
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Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery.
mRNA has broad potential as a therapeutic. Current clinical efforts are focused on vaccination, protein replacement therapies, and treatment of genetic diseases. The clinical translation of mRNA therapeutics has been made possible through advances in the design of mRNA manufacturing and intracellular delivery methods. However, broad application of mRNA is still limited by the need for improved delivery systems. In this review, we discuss the challenges for clinical translation of mRNA-based therapeutics, with an emphasis on recent advances in biomaterials and delivery strategies, and we present an overview of the applications of mRNA-based delivery for protein therapy, gene editing, and vaccination.
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Near-infrared optogenetic engineering of photothermal nanoCRISPR for programmable genome editing.
We herein report an optogenetically activatable CRISPR-Cas9 nanosystem for programmable genome editing in the second near-infrared (NIR-II) optical window. The nanosystem, termed nanoCRISPR, is composed of a cationic polymer-coated Au nanorod (APC) and Cas9 plasmid driven by a heat-inducible promoter. The APC not only serves as a carrier for intracellular plasmid delivery but also can harvest external NIR-II photonic energy and convert it into local heat to induce the gene expression of the Cas9 endonuclease. Due to high transfection activity, the APC shows strong ability to induce a significant level of disruption in different genomic loci upon optogenetic activation. Moreover, the precise control of genome-editing activity can be simply programmed by finely tuning exposure time and irradiation time in vitro and in vivo and also enables editing at multiple time points, thus proving the sensitivity and inducibility of such an editing modality. The NIR-II optical feature of nanoCRISPR enables therapeutic genome editing at deep tissue, by which treatment of deep tumor and rescue of fulminant hepatic failure are demonstrated as proof-of-concept therapeutic examples. Importantly, this modality of optogenetic genome editing can significantly minimize the off-target effect of CRISPR-Cas9 in most potential off-target sites. The optogenetically activatable CRISPR-Cas9 nanosystem we have developed offers a useful tool to expand the current applications of CRISPR-Cas9, and also defines a programmable genome-editing strategy toward high precision and spatial specificity.
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An RNAi suppressor activates in planta virus-mediated gene editing.
RNA-guided CRISPR/Cas9 technology has been developed for gene/genome editing (GE) in organisms across kingdoms. However, in planta delivery of the two core GE components, Cas9 and small guide RNA (sgRNA), often involves time-consuming and labor-intensive production of transgenic plants. Here we show that Foxtail mosaic virus, a monocot- and dicot-infecting potexvirus, can simultaneously express Cas9, sgRNA, and an RNAi suppressor to efficiently induce GE in Nicotiana benthamiana through a transgenic plant-free manner.
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Synergistic strategies for glioblastoma treatment: CRISPR-based multigene editing combined with immune checkpoint blockade.
Glioblastoma (GBM) is a primary brain tumor known for its high levels of aggressiveness and resistance to current treatments such as radiotherapy and chemotherapy. As a result, there is a pressing need for innovative therapeutic approaches to combat GBM. Thus, we have developed an engineered multifunctional extracellular vesicle (EV) delivery system that offers an "all-in-one" strategy for GBM therapy. Our approach involved the use of genetic engineering to the long-lasting production of PD-1 and the brain-specific peptide angiopep-2 on the surface of EVs. These modified EVs were then utilized to rejuvenate exhausted CD8+ T cells blocking PD-L1, resulting in significant therapeutic benefits for GBM treatment. Furthermore, the EVs contained Cas9 protein and sgRNA for precise and minimally invasive gene therapy, which addressing the key barriers associated with in vivo CRISPR‒Cas9 gene editing treatment. The multigene editing of EVs resulted in efficient intratumor multisite gene editing (PLK1: 58.6%, VEGF: 52.7%), leading to the successful apoptosis of tumor cells in vivo and demonstrating an antiangiogenic effect. This research introduces a promising universal platform for combining immune checkpoint blockade therapy with gene editing treatment.
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Pioneering the future: CRISPR-Cas9 gene therapy for hereditary hemorrhagic telangiectasia.
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[CRISPR-CAS9. The greatest advancement in genetic edition techniques requires an ethical reflection].
The adaptation of the CRISPR system as a genetic editing tool has led to a revolution in many fields of application, as this technique is considerably faster, easier to perform and more efficient than predecessor techniques. However, some of these applications raise objective ethical issues that must be addressed. In this paper we discuss, based on the most recent data, the different issues related to CRISPR applications on the germ line, its introduction in clinical trials, the genetic edition of animals and plants for human consumption and the novel gene drive.
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CRISPRedict: a CRISPR-Cas9 web tool for interpretable efficiency predictions.
The development of the CRISPR-Cas9 technology has provided a simple yet powerful system for genome editing. Current gRNA design tools serve as an important platform for the efficient application of the CRISPR systems. However, most of the existing tools are black-box models that suffer from limitations, such as variable performance and unclear mechanism of decision making. Here, we introduce CRISPRedict, an interpretable gRNA efficiency prediction model for CRISPR-Cas9 gene editing. Its strength lies in the fact that it can accurately predict efficient guide RNAs-with equivalent performance to state-of-the-art tools-while being a simple linear model. Implemented as a user-friendly web server, CRISPRedict offers (i) quick and accurate predictions across various experimental conditions (e.g. U6/T7 transcription); (ii) regression and classification models for scoring gRNAs and (iii) multiple visualizations to explain the obtained results. Given its performance, interpretability, and versatility, we expect that it will assist researchers in the gRNA design process and facilitate genome editing research. CRISPRedict is available for use at http://www.crispredict.org/.
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