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Found: 26 174
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CRISPR-associated nucleases: the Dawn of a new age of efficient crop improvement.
The world stands at a new threshold today. As a planet, we face various challenges, and the key one is how to continue to produce enough food, feed, fiber, and fuel to support the burgeoning population. In the past, plant breeding and the ability to genetically engineer crops contributed to increasing food production. However, both approaches rely on random mixing or integration of genes, and the process can be unpredictable and time-consuming. Given the challenge of limited availability of natural resources and changing environmental conditions, the need to rapidly and precisely improve crops has become urgent. The discovery of CRISPR-associated endonucleases offers a precise yet versatile platform for rapid crop improvement. This review summarizes a brief history of the discovery of CRISPR-associated nucleases and their application in genome editing of various plant species. Also provided is an overview of several new endonucleases reported recently, which can be utilized for editing of specific genes in plants through various forms of DNA sequence alteration. Genome editing, with its ever-expanding toolset, increased efficiency, and its potential integration with the emerging synthetic biology approaches hold promise for efficient crop improvement to meet the challenge of supporting the needs of future generations.
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Functional Characterization of Novel U6 RNA Polymerase III Promoters: Their Implication for CRISPR-Cas9-Mediated Gene Editing in Aspergillus oryzae.
U6 RNA polymerase III promoter (PU6), which is a key element in controlling the generation of single-guide RNA (sgRNA) for gene editing through CRISPR-Cas9 system, was investigated in this work. Using bioinformatics approach, two novel U6 ribonucleic acid (U6 RNA) sequences of Aspergillus niger were identified, showing that they had conserved motifs similar to other U6 RNAs. The putative PU6 located at the upstream sequence of A. niger U6 RNA exhibited the consensus motif, CCAATYA, and the TATA box which shared highly conserved characteristics across Aspergilli, whereas the A- and B-boxes were found at the intragenic and downstream of the structural genes, respectively. Using Aspergillus oryzae as a workhorse system, the function of A. niger PU6s for controlling the transcripts of sgRNA was verified, in which the orotidine-5'-phosphate decarboxylase (pyrG) sequence was used as a target for gene disruption through CRISPR-Cas9 system. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis of the selected pyrG auxotrophic strains showed the expression of sgRNA, indicating that the non-native promoters could efficiently drive sgRNA expression in A. oryzae. These identified promoters are useful genetic tools for precise engineering of metabolic pathways in the industrially important fungus through the empowered CRISPR-Cas9-associated gene-editing system.
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Gene Edited Tumor Cells Kill and Prevent Cancer in Mice.
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New horizons in reproductive biology: a special issue.
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Primary B cell engineering for therapeutic research.
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CRISPR-Cas-mediated transcriptional modulation: The therapeutic promises of CRISPRa and CRISPRi.
The CRISPR-Cas system is commonly known for its ability to cleave DNA in a programmable manner, which has democratized gene editing and facilitated recent breakthroughs in gene therapy. However, newer iterations of the technology using nuclease-disabled Cas enzymes have spurred a variety of different types of genetic engineering platforms such as transcriptional modulation using the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems. This review introduces the creation of these programmable transcriptional modulators, various methods of delivery utilized for these systems, and recent technological developments. CRISPRa and CRISPRi have also been implemented in genetic screens for interrogating gene function and discovering genes involved in various biological pathways. We describe recent compelling examples of how these tools have become powerful means to unravel genetic networks and uncovering important information about devastating diseases. Finally, we provide an overview of preclinical studies in which transcriptional modulation has been used therapeutically, and we discuss potential future directions of these novel modalities.
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Comprehensive strategies for constructing efficient CRISPR/Cas based cancer therapy: Target gene selection, sgRNA optimization, delivery methods and evaluation.
Cancer is a complicated disease that results from the interplay between specific changes in cellular genetics and diverse microenvironments. The application of high-performance and customizable clustered regularly interspaced palindromic repeats/associated protein (CRISPR/Cas) nuclease systems has significantly enhanced genome editing for accurate cancer modeling and facilitated simultaneous genetic modification for cancer therapy and mutation identification. Achieving an effective CRISPR/Cas platform for cancer treatment depends on the identification, selection, and optimization of specific mutated genes in targeted cancer tissues. However, overcoming the off-target effects, specificity, and immunogenicity are additional challenges that must be addressed while developing a gene editing system for cancer therapy. From this perspective, we briefly covered the pipeline of CRISPR/Cas cancer therapy, identified target genes to optimize gRNAs and sgRNAs, and explored alternative delivery modalities, including viral, non-viral, and extracellular vesicles. In addition, the list of patents and current clinical trials related to this unique cancer therapy method is discussed. In summary, we have discussed comprehensive start-to-end pipeline strategies for CRISPR/Cas development to advance the precision, effectiveness, and safety of clinical applications for cancer therapy.
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Simulating the Commercial Implementation of Gene-Editing for Influenza A Virus Resistance in Pigs: An Economic and Genetic Analysis.
The development of swine Influenza A Virus resistance along with genetic technologies could complement current control measures to help to improve animal welfare standards and the economic efficiency of pig production. We have created a simulation model to assess the genetic and economic implications of various gene-editing methods that could be implemented in a commercial, multi-tiered swine breeding system. Our results demonstrate the length of the gene-editing program was negatively associated with genetic progress in commercial pigs and that the time required to reach fixation of resistance alleles was reduced if the efficiency of gene-editing is greater. The simulations included the resistance conferred in a digenic model, the inclusion of genetic mosaicism in progeny, and the effects of selection accuracy. In all scenarios, the level of mosaicism had a greater effect on the time required to reach resistance allele fixation and the genetic progress of the herd than gene-editing efficiency and zygote survival. The economic analysis highlights that selection accuracy will not affect the duration of gene-editing and the investment required compared to the effects of gene-editing-associated mosaicism and the swine Influenza A Virus control strategy on farms. These modelling results provide novel insights into the economic and genetic implications of targeting two genes in a commercial pig gene-editing program and the effects of selection accuracy and mosaicism.
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Coordinated gene upregulation in maize through CRISPR/Cas-mediated enhancer insertion.
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Construction of Homozygous Mutants of Migratory Locust using CRISPR/Cas9 Technology.
The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture, preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.
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