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Off-target Cas9 crystallized.
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A Conversation with Jennifer Doudna.
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Targeted Insertion in Nicotiana benthamiana Genomes via Protoplast Regeneration.
Insertion of a specific sequence in a targeted region for precise editing is still a major challenge in plants. Current protocols rely on inefficient homology-directed repair or non-homologous end-joining with modified double-stranded oligodeoxyribonucleotides (dsODNs) as donors. We developed a simple protocol that eliminates the need for expensive equipment, chemicals, modifications of donor DNA, and complicated vector construction. The protocol uses polyethylene glycol (PEG)-calcium to deliver low-cost, unmodified single-stranded oligodeoxyribonucleotides (ssODNs) and CRISPR/Cas9 ribonucleoprotein (RNP) complexes into Nicotiana benthamiana protoplasts. Regenerated plants were obtained from edited protoplasts with an editing frequency of up to 50% at the target locus. The inserted sequence was inherited to the next generation; this method thus opens the possibility for the future exploration of genomes by targeted insertion in plants.
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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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Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects.
Several existing technologies enable short genomic alterations including generating indels and short nucleotide variants, however, engineering more significant genomic changes is more challenging due to reduced efficiency and precision. Here, we developed RecT Editor via Designer-Cas9-Initiated Targeting (REDIT), which leverages phage single-stranded DNA-annealing proteins (SSAP) RecT for mammalian genome engineering. Relative to Cas9-mediated homology-directed repair (HDR), REDIT yielded up to a 5-fold increase of efficiency to insert kilobase-scale exogenous sequences at defined genomic regions. We validated our REDIT approach using different formats and lengths of knock-in templates. We further demonstrated that REDIT tools using Cas9 nickase have efficient gene-editing activities and reduced off-target errors, measured using a combination of targeted sequencing, genome-wide indel, and insertion mapping assays. Our experiments inhibiting repair enzyme activities suggested that REDIT has the potential to overcome limitations of endogenous DNA repair steps. Finally, our REDIT method is applicable across cell types including human stem cells, and is generalizable to different Cas9 enzymes.
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Recovery of the non-functional EGFP-assisted identification of mutants generated by CRISPR/Cas9.
The recovery of non-functional-enhanced green fluorescence protein can be used as indicator to facilitate the identification of mutants generated by CRISPR/Cas9. The CRISPR/Cas9 system is a powerful tool for genome editing and it has been employed to knock out genes of interest in multiple plant species. Identification of desired mutants from regenerated plants is necessary prior to functional study. Current screening methods work based on the purification of genomic DNA and it would be laborious and time consuming using these methods to screen mutants from a large population of seedlings. Here, we developed the non-functional enhanced green fluorescence protein (nEGFP) reporter gene by inserting a single guide RNA (sgRNA) and the protospacer adjacent motif in the 5' coding region of EGFP, and the activity of nEGFP could be recovered after successful targeted editing. Using the nEGFP as the reporter gene in Nicotiana tabacum, we found that over 94% of the plants exhibiting EGFP fluorescence were confirmed to be desired mutants. The use of this nEGFP reporter construct had limited negative effect on editing efficiency, and the expression of Cas9 and sgRNA was not affected. Moreover, this method was also applied in grape by targeting the phytoene desaturase gene (PDS), and the grape cells with EGFP signal were revealed to contain targeted mutations in VvPDS. Our results show that the nEGFP gene can be used as reporter to help screen mutants according to the recovered EGFP fluorescence during the application of CRISPR/Cas9 in plants.
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Map of ethical conflicts of the CRISPR-Cas9 gene edition technique.
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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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New hope and promise with CRISPR-Cas9 technology for the treatment of HIV.
The commencement of Highly Active Antiretroviral Therapy almost completely stopped viral replication, enabling the immune system to restore its full functionality. The rise in life expectancy has resulted in a decrease in the incidence of classical infections and HIV-associated cancers. HAART has raised concerns, including its exorbitant cost (which hinders its implementation in developing nations), the need for strict adherence, and the potential for both immediate and prolonged ill effects. Lipodystrophy is a significant long-term consequence of HIV that may result in central fat accumulation and severe peripheral fat depletion. Current initiatives to tackle these difficulties include the global expansion of access to HAART, the development of novel drugs that mitigate early side effects, and the introduction of once-daily drug combinations that enhance adherence. The CRISPR-Cas9 system has facilitated the creation of a powerful instrument for precise gene editing. This method has lately established itself as the gold standard for efficient HIV-1 genome editing in HIV therapy, owing to progress in related disciplines. CRISPR may be customized to cleave specific sequences by altering Cas9. This article offers a concise overview of promising CRISPR-Cas9 technology. This technique has the potential to halt the transmission of HIV-1 and alleviate its symptoms. CRISPR-Cas9 technology will be significant in the fight against HIV-1 in the future.
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Controversial CRISPR 'gene drives' tested in mammals for the first time.
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