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Gene Editing for Treatment of Neurological Infections.
The study of neurological infections by viruses defines the field of neurovirology, which has emerged in the last 30 years and was founded upon the discovery of a number of viruses capable of infecting the human nervous system. Studies have focused on the molecular and biological basis of viral neurological diseases with the aim of revealing new therapeutic options. The first studies of neurovirological infections can be traced back to the discovery that some viruses have an affinity for the nervous system with research into rabies by Louis Pasteur and others in the 1880s. Today, the immense public health impact of neurovirological infections is illustrated by diseases such as neuroAIDS, progressive multifocal leukoencephalopathy, and viral encephalitis. Recent research has seen the development of powerful new techniques for gene editing that promise revolutionary opportunities for the development of novel therapeutic options. In particular, clustered regulatory interspaced short palindromic repeat-associated 9 system provides an effective, highly specific and versatile tool for targeting DNA viruses that are beginning to allow the development of such new approaches. In this short review, we discuss these recent developments, how they pertain to neurological infections, and future prospects.
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Mass spectrometric analysis of purine de novo biosynthesis intermediates.
Purines are essential molecules for all forms of life. In addition to constituting a backbone of DNA and RNA, purines play roles in many metabolic pathways, such as energy utilization, regulation of enzyme activity, and cell signaling. The supply of purines is provided by two pathways: the salvage pathway and de novo synthesis. Although purine de novo synthesis (PDNS) activity varies during the cell cycle, this pathway represents an important source of purines, especially for rapidly dividing cells. A method for the detailed study of PDNS is lacking for analytical reasons (sensitivity) and because of the commercial unavailability of the compounds. The aim was to fully describe the mass spectrometric fragmentation behavior of newly synthesized PDNS-related metabolites and develop an analytical method. Except for four initial ribotide PDNS intermediates that preferentially lost water or phosphate or cleaved the forming base of the purine ring, all the other metabolites studied cleaved the glycosidic bond in the first fragmentation stage. Fragmentation was possible in the third to sixth stages. A liquid chromatography-high-resolution mass spectrometric method was developed and applied in the analysis of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual enzymatic steps of PDNS and the salvage pathway. The identities of the newly synthesized intermediates of PDNS were confirmed by comparing the fragmentation patterns of the synthesized metabolites with those produced by cells (formed under pathological conditions of known and theoretically possible defects of PDNS). The use of stable isotope incorporation allowed the confirmation of fragmentation mechanisms and provided data for future fluxomic experiments. This method may find uses in the diagnosis of PDNS disorders, the investigation of purinosome formation, cancer research, enzyme inhibition studies, and other applications.
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SAA-Cas9: A tunable genome editing system with increased bio-safety and reduced off-target effects.
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Beyond Viruses: Transposons and CRISPR-Programmable Nonviral Systems for Genome Engineering.
Human gene therapy has been transformed by repurposing mobile genetic elements as tools for efficient gene delivery and precise genome modification. Viruses, transposons, and retrotransposons mobilize genetic material through distinct mechanisms with distinct advantages for genome engineering platforms with therapeutic potential. This review describes the mechanisms, performance characteristics, and translational relevance of nonviral methods of gene transfer and genome engineering, with emphasis on engineered systems such as the Sleeping Beauty (SB) and piggyBac (PB) DNA transposons, R2 retrotransposon-based platforms, CRISPR-associated transposons (CASTs), and prime editing (PE). Clinical trials using both SB- and PB-engineered cells have shown feasibility by demonstrating sustained therapeutic effect through stable transgene integration, such as CD19-directed CAR-T cell therapy for hematological malignancies. These transposon-based vectors enable robust gene transfer for cell engineering, but are fairly promiscuous with respect to where in the genome they insert their genetic cargo. On the contrary, R2-derived systems and CASTs represent promising technologies for programmable insertion, but are still under preclinical development. CRISPR-based PE provides a distinct route to precise sequence correction without double-strand breaks and is particularly attractive for the correction of monogenic disorders. Taken together, these technologies define a continuum from natural genome mobility to clinically relevant genome engineering. Continued optimization of delivery, targeting, and editing fidelity will determine how rapidly these approaches move from experimental innovation into routine clinical use.
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CRISPR-Mediated Genome Editing and Gene Repression in Scheffersomyces stipitis.
Scheffersomyces stipitis, renowned for its native xylose-utilizing capacity, has recently demonstrated its potential in producing health-promoting shikimate pathway derivatives. However, its broader application is hampered by the low transformation efficiency and the lack of genetic engineering tools to enable sophisticated genomic manipulations. S. stipitis employs the predominant non-homologous end joining (NHEJ) mechanism for repairing DNA double-strand breaks (DSB), which is less desired due to its incompetence in achieving precise genome editing. Using CRISPR technology, here a ku70Δku80Δ deficient strain in which homologous recombination (HR)-based genome editing appeared dominant for the first time in S. stipitis is constructed. To build all essential tools for efficiently manipulating this highly promising nonconventional microbial host, the gene knockdown tool is also established, and repression efficiency is improved by incorporating a transcriptional repressor Mxi1 into the CRISPR-dCas9 platform. All these results are obtained with the improved transformation efficiency, which is 191-fold higher than that obtained with the traditional parameters used in yeast transformation. This work paves the way for advancing a new microbial chassis and provides a guideline for developing efficient CRISPR tools in other nonconventional yeasts.
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Selective targeting of the oncogenic KRAS G12S mutant allele by CRISPR/Cas9 induces efficient tumor regression.
Rationale: KRAS is one of the most frequently mutated oncogenes in cancers. The protein's picomolar affinity for GTP/GDP and smooth protein structure resulting in the absence of known allosteric regulatory sites makes its genomic-level activating mutations a difficult but attractive target. Methods: Two CRISPR systems, genome-editing CRISPR/SpCas9 and transcription-regulating dCas9-KRAB, were developed to deplete the KRAS G12S mutant allele or repress its transcription, respectively, with the goal of treating KRAS-driven cancers. Results: SpCas9 and dCas9-KRAB systems with a sgRNA targeting the mutant allele blocked the expression of the mutant KRAS gene, leading to an inhibition of cancer cell proliferation. Local adenoviral injections using SpCas9 and dCas9-KRAB systems suppressed tumor growth in vivo. The gene-depletion system (SpCas9) performed more effectively than the transcription-suppressing system (dCas9-KRAB) on tumor inhibition. Application of both Cas9 systems to wild-type KRAS tumors did not affect cell proliferation. Furthermore, through bioinformatic analysis of 31555 SNP mutations of the top 20 cancer driver genes, the data showed that our mutant-specific editing strategy could be extended to a reference list of oncogenic mutations with high editing potentials. This pipeline could be applied to analyze the distribution of PAM sequences and survey the best alternative targets for gene editing. Conclusion: We successfully developed both gene-depletion and transcription-suppressing systems to specifically target an oncogenic KRAS mutant allele that led to significant tumor regression. These findings show the potential of CRISPR-based strategies for the treatment of tumors with driver gene mutations.
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First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill.
Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the phytoene desaturase gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.
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Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response.
CRISPR/Cas9-mediated gene editing of stem cells and primary cell types has several limitations for clinical applications. The direct delivery of ribonucleoprotein (RNP) complexes consisting of Cas9 nuclease and guide RNA (gRNA) has improved DNA- and virus-free gene modifications, but it does not enable the essential enrichment of the gene-edited cells. Here, we established a protocol for the fluorescent labeling and delivery of CRISPR/Cas9-gRNA RNP in primary human hematopoietic stem and progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). As a proof of principle for genes with low-abundance transcripts and context-dependent inducible expression, we successfully deleted growth arrest and DNA-damage-inducible β (GADD45B). We found that GADD45B is indispensable for DNA damage protection and survival in stem cells. Thus, we describe an easy and efficient protocol of DNA-free gene editing of hard-to-target transcripts and enrichment of gene-modified cells that are generally difficult to transfect.
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Current therapies for osteoarthritis and prospects of CRISPR-based genome, epigenome, and RNA editing in osteoarthritis treatment.
Osteoarthritis (OA) is one of the most common degenerative joint diseases worldwide, causing pain, disability, and decreased quality of life. The balance between regeneration and inflammation-induced degradation results in multiple etiologies and complex pathogenesis of OA. Currently, there is a lack of effective therapeutic strategies for OA treatment. With the development of CRISPR-based genome, epigenome, and RNA editing tools, OA treatment has been improved by targeting genetic risk factors, activating chondrogenic elements, and modulating inflammatory regulators. Supported by cell therapy and in vivo delivery vectors, genome, epigenome, and RNA editing tools may provide a promising approach for personalized OA therapy. This review summarizes CRISPR-based genome, epigenome, and RNA editing tools that can be applied to the treatment of OA and provides insights into the development of CRISPR-based therapeutics for OA treatment. Moreover, in-depth evaluations of the efficacy and safety of these tools in human OA treatment are needed.
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A rocky road for the maturation of embryo-editing methods.
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