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Elimination of editing plasmid mediated by theophylline riboswitch in Zymomonas mobilis.
Zymomonas mobilis is regarded as a potential chassis for the production of platform chemicals. Genome editing using the CRISPR-Cas system could meet the need for gene modification in metabolic engineering. However, the low curing efficiency of CRISPR editing plasmid is a common bottleneck in Z. mobilis. In this study, we utilized a theophylline-dependent riboswitch to regulate the expression of the replicase gene of the editing plasmid, thereby promoting the elimination of exogeneous plasmid. The riboswitch D (RSD) with rigorous regulatory ability was identified as the optimal candidate by comparing the transformation efficiency of four theophylline riboswitch-based backbone editing plasmids, and the optimal theophylline concentration for inducing RSD was determined to be 2 mM. A highly effective method for eliminating the editing plasmid, cells with RSD-based editing plasmid which were cultured in liquid and solid RM media in alternating passages at 37 °C without shaking, was established by testing the curing efficiency of backbone editing plasmids pMini and pMini-RSD in RM medium with or without theophylline at 30 °C or 37 °C. Finally, the RSD-based editing plasmid was applied to genome editing, resulting in an increase of more than 10% in plasmid elimination efficiency compared to that of pMini-based editing plasmid. KEY POINTS: • An effective strategy for curing CRISPR editing plasmid has been established in Z. mobilis. • Elimination efficiency of the CRISPR editing plasmid was enhanced by 10% to 20% under the regulation of theophylline-dependent riboswitch RSD.
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New models: Gene-editing boom means changing landscape for primate work.
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Phenotypic novelty by CRISPR in plants.
Genome editing by CRISPR is now routinely used in plant biology for unravelling gene functions and improving agronomical traits. CRISPR opens up the possibility of genome manipulations which would have been unthinkable a few years ago. In this perspective, we discuss and suggest CRISPR-mediated approaches for steering plant development, also highlighting potential challenges.
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Chemical Modifications in Nucleic Acid Therapeutics.
Nucleic acid-based therapies (NATs) have become an increasingly prominent class of drugs due to the recent clinical successes made possible by nucleic acid chemical modifications. This class of therapies includes reagents that inhibit gene expression (antisense oligonucleotides (ASOs) or RNA interference (RNAi)), modulate gene structure (splice-shifting ASOs), increase protein expression (messenger RNA (mRNA)) or direct specific editing of the mammalian genome (CRISPR/Cas gene editing). Each of these technologies relies on specific combinations of chemically modified nucleic acids to increase drug efficacy, safety, and uptake efficiency in desired cell types. The knowledge gained from years of characterizing the biochemical properties of chemically modified oligonucleotides (ONs) combined with recent regulatory approvals will hopefully accelerate more NATs into the clinic to treat currently undruggable or ultrarare diseases. This review discusses the most employed chemical modifications in each of the aforementioned nucleic acid-based technologies and provides an overview of select publications that have demonstrated milestones and successes in improving ON efficacy and/or mitigating undesired off-target effects. Key innovations in chemical modifications that are expanding clinical capabilities are highlighted, casting a positive light on the future of nucleic acid medicine.
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A cut above: pair that developed CRISPR earns historic award.
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The Potential of CRISPR/Cas9 in Hematotherapy.
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Regulatory aspects of gene editing in Argentina.
Argentina is a world leader in regards to regulation and adoption of genetically modified (GM) crops. As a consequence, the regulatory aspects of gene editing applied to agriculture were considered proactively by the Argentinian regulators, who implemented simple but solid pioneering regulatory criteria for gene edited crops. At present, the Argentine regulatory system is fully able to establish if a gene-edited crop should be classified (and handled) either as a GM crop or a conventional new variety. To this end, the concept of "novel combination of genetic material" derived from the Cartagena Protocol on Biosafety is of decisive importance. After some pilot cases that have been managed under this criteria, now applicants appreciate the ease, speed and predictability of the regulation. Moreover, it has been considered by other countries in the course of developing their own regulations, thus acting also as a harmonization factor for the safe and effective insertion of these technologies in the global market.
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Visualized Multigene Editing System for Aspergillus niger.
The resistance markers could ensure the entry of the CRISPR/Cas9 system into Aspergillus niger cells instead of gene editing. To increase the efficiency of positive colony screening on the primary transformation plates, we designed a visualized multigene editing system (VMS) via a unique tRNA-guide RNA (gRNA) array containing the gRNAs of a pigment gene albA and target genes. Disruption of albA produces white colonies, and the sequences of the endogenous tRNAAla, tRNAPhe, tRNAArg, tRNAIle, and tRNALeu enhance gRNA release. The disruption efficiencies of multigene were analyzed in the A. niger strain AG11 using ammA, amyA, prtT, kusA, and glaA as reporters. In white colonies on the primary transformation plates, the disruption rates of one-, two-, three-, four-, and five-target genes reached 89.2, 70.91, 50, 22.41, and 4.17%, respectively. The VMS developed here provides an effective method for screening homokaryotic multigene editing strains of A. niger.
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A Recombinant Targeted Bionanoparticle In Vitro Expressing a Gene-Editing Protein for Epidermal Growth Factor Receptor Mutant Lung Cancer.
Epidermal growth factor receptor (EGFR) mutation is a significant driving factor in the occurrence and progression of lung cancer. How to effectively block the abnormal expression of EGFR remains a key issue that urgently needs to be addressed, as it is of vital importance for the effective treatment of this cancer. Here, we report a recombinant targeted bionanoparticle that in vitro expresses Cas9 protein (tBioNP vitro-Cas9) for gene editing of EGFR mutant lung cancer. The nanoparticle was developed by transfection of four plasmids (Gag-Cas9, Gag, sgRNA, VSV-G Azi) into 293T cells to form a type of bionanoparticle and modifying it with a targeted polymer material (DBCO-PEG-FA), and it showed a cancer-targeted property, faster cancer cellular uptake, higher gene editing efficiency with lower off-target effects, and therapy efficacy in mice, indicating a translational prospect. In conclusion, the study provides a recombinant bionanoparticle in vitro expressing a Cas9 gene editing system and offers a potential strategy for gene therapy of EGFR mutant lung cancer.
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WheatCRISPR: a web-based guide RNA design tool for CRISPR/Cas9-mediated genome editing in wheat.
BACKGROUND: CRISPR/Cas9 gene editing has become a revolutionary technique for crop improvement as it can facilitate fast and efficient genetic changes without the retention of transgene components in the final plant line. Lack of robust bioinformatics tools to facilitate the design of highly specific functional guide RNAs (gRNAs) and prediction of off-target sites in wheat is currently an obstacle to effective application of CRISPR technology to wheat improvement. DESCRIPTION: We have developed a web-based bioinformatics tool to design specific gRNAs for genome editing and transcriptional regulation of gene expression in wheat. A collaborative study between the Broad Institute and Microsoft Research used large-scale empirical evidence to devise algorithms (Doech et al., 2016, Nature Biotechnology 34, 184-191) for predicting the on-target activity and off-target potential of CRISPR/SpCas9 (Streptococcus pyogenes Cas9). We applied these prediction models to determine on-target specificity and potential off-target activity for individual gRNAs targeting specific loci in the wheat genome. The genome-wide gRNA mappings and the corresponding Doench scores predictive of the on-target and off-target activities were used to create a gRNA database which was used as a data source for the web application termed WheatCRISPR. CONCLUSION: The WheatCRISPR tool allows researchers to browse all possible gRNAs targeting a gene or sequence of interest and select effective gRNAs based on their predicted high on-target and low off-target activity scores, as well as other characteristics such as position within the targeted gene. It is publicly available at https://crispr.bioinfo.nrc.ca/WheatCrispr/ .
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