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Fostering a prevention mindset for responsible gene editing.
In this special issue, we solicited three commentators to discuss issues specific to the responsible conduct of research as it relates to the birth of gene-edited children. We explore the ethics of prevention and its ties to the responsible conduct of gene-editing research to introduce three commentaries addressing: (1) the relevance of cultural and regulatory context in China, (2) how to actualize calls for greater public engagement, and (3) where we might improve graduate education of genomic researchers.
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Advances in bread wheat production through CRISPR/Cas9 technology: a comprehensive review of quality and other aspects.
This review provides a comprehensive overview of the CRISPR/Cas9 technique and the research areas of this gene editing tool in improving wheat quality. Wheat (Triticum aestivum L.), the basic nutrition for most of the human population, contributes 20% of the daily energy needed because of its, carbohydrate, essential amino acids, minerals, protein, and vitamin content. Wheat varieties that produce high yields and have enhanced nutritional quality will be required to fulfill future demands. Hexaploid wheat has A, B, and D genomes and includes three like but not identical copies of genes that influence important yield and quality. CRISPR/Cas9, which allows multiplex genome editing provides major opportunities in genome editing studies of plants, especially complicated genomes such as wheat. In this overview, we discuss the CRISPR/Cas9 technique, which is credited with bringing about a paradigm shift in genome editing studies. We also provide a summary of recent research utilizing CRISPR/Cas9 to investigate yield, quality, resistance to biotic/abiotic stress, and hybrid seed production. In addition, we provide a synopsis of the laboratory experience-based solution alternatives as well as the potential obstacles for wheat CRISPR studies. Although wheat's extensive genome and complicated polyploid structure previously slowed wheat genetic engineering and breeding progress, effective CRISPR/Cas9 systems are now successfully used to boost wheat development.
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Molecular biology at the cutting edge: A review on CRISPR/CAS9 gene editing for undergraduates.
Disrupting a gene to determine its effect on an organism's phenotype is an indispensable tool in molecular biology. Such techniques are critical for understanding how a gene product contributes to the development and cellular identity of organisms. The explosion of genomic sequencing technologies combined with recent advances in genome-editing techniques has elevated the possibilities of genetic manipulations in numerous organisms in which these experiments were previously not readily accessible or possible. Introducing the next generation of molecular biologists to these emerging techniques is key in the modern biology classroom. This comprehensive review introduces undergraduates to CRISPR/Cas9 editing and its uses in genetic studies. The goals of this review are to explain how CRISPR functions as a prokaryotic immune system, describe how researchers generate mutations with CRISPR/Cas9, highlight how Cas9 has been adapted for new functions, and discuss ethical considerations of genome editing. Additionally, anticipatory guides and questions for discussion are posed throughout the review to encourage active exploration of these topics in the classroom. Finally, the supplement includes a study guide and practical suggestions to incorporate CRISPR/Cas9 experiments into lab courses at the undergraduate level. © 2018 The Authors Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 46(2):195-205, 2018.
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An NGS-based approach for precise and footprint-free CRISPR-based gene editing in human stem cells.
Precise gene editing with conventional CRISPR/Cas9 is often constrained by low knock-in (KI) efficiencies (≈ 2-20 %) in human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). This limitation typically necessitates labour-intensive manual isolation and genotyping of hundreds of colonies to identify correctly edited cells. Fluorescence- or antibiotic-based enrichment methods facilitate the identification process but can compromise cell viability and genomic integrity. Here, we present a footprint-free editing strategy that combines low-density seeding with next-generation sequencing (NGS) to rapidly identify cell populations containing precisely modified clones. By optimising the transfection workflow and adhering to CRISPR/Cas9 KI design principles, we achieved high average editing efficiencies of 64 % in hiPSCs (introducing a Brugada syndrome-associated variant) and 51 % in hESCs (introducing a neurodevelopmental disorder (NDD)-associated variant). Furthermore, under suboptimal CRISPR design conditions, this approach successfully identified hESC clones carrying a second NDD-associated variant, despite average KI efficiencies below 1 %. Importantly, genomic integrity was preserved throughout subcloning rounds, as confirmed by Sanger sequencing and single nucleotide polymorphism (SNP) array analysis. Hence, this NGS-based enrichment strategy reliably identifies desired KI clones under both optimal and challenging conditions, reducing the need for extensive colony screening and offering an effective alternative to fluorescence- and antibiotic-based selection methods.
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Two stones, one bird: co-targeting facilitates precise gene editing of non-selectable genes in Chlamydomonas.
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Recent advances in the application of multiplex genome editing in Saccharomyces cerevisiae.
Saccharomyces cerevisiae is a widely used microorganism and a greatly popular cell factory for the production of various chemicals. In order to improve the yield of target chemicals, it is often necessary to increase the copy numbers of key genes or engineer the related metabolic pathways, which traditionally required time-consuming repetitive rounds of gene editing. With the development of gene-editing technologies such as meganucleases, TALENs, and the CRISPR/Cas system, multiplex genome editing has entered a period of rapid development to speed up cell factory optimization. Multi-copy insertion and removing bottlenecks in biosynthetic pathways can be achieved through gene integration and knockout, for which multiplexing can be accomplished by targeting repetitive sequences and multiple sites, respectively. Importantly, the development of the CRISPR/Cas system has greatly increased the speed and efficiency of multiplex editing. In this review, the various multiplex genome editing technologies in S. cerevisiae were summarized, and the principles, advantages, and the disadvantages were analyzed and discussed. Finally, the practical applications and future prospects of multiplex genome editing were discussed. KEY POINTS: • The development of multiplex genome editing in S. cerevisiae was summarized. • The pros and cons of various multiplex genome editing technologies are discussed. • Further prospects on the improvement of multiplex genome editing are proposed.
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A critical review on therapeutic approaches of CRISPR-Cas9 in diabetes mellitus.
Diabetes mellitus (D.M.) is a common metabolic disorder caused mainly by combining two primary factors, which are (1) defects in insulin production by the pancreatic β-cells and (2) responsiveness of insulin-sensitive tissues towards insulin. Despite the rapid advancement in medicine to suppress elevated blood glucose levels (hyperglycemia) and insulin resistance associated with this hazard, a demand has undoubtedly emerged to find more effective and curative dimensions in therapeutic approaches against D.M. The administration of diabetes treatment that emphasizes insulin production and sensitivity may result in unfavorable side effects, reduced adherence, and potential treatment ineffectiveness. Recent progressions in genome editing technologies, for instance, in zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR-Cas)-associated nucleases, have greatly influenced the gene editing technology from concepts to clinical practices. Improvements in genome editing technologies have also opened up the possibility to target and modify specific genome sequences in a cell directly. CRISPR/Cas9 has proven effective in utilizing ex vivo gene editing in embryonic stem cells and stem cells derived from patients. This application has facilitated the exploration of pancreatic beta-cell development and function. Furthermore, CRISPR/Cas9 enables the creation of innovative animal models for diabetes and assesses the effectiveness of different therapeutic strategies in treating the condition. We, therefore, present a critical review of the therapeutic approaches of the genome editing tool CRISPR-Cas9 in treating D.M., discussing the challenges and limitations of implementing this technology.
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Induced Pluripotent Stem Cells (iPSCs) and Gene Therapy: A New Era for the Treatment of Neurological Diseases.
To date, gene therapy has employed viral vectors to deliver therapeutic genes. However, recent progress in molecular and cell biology has revolutionized the field of stem cells and gene therapy. A few years ago, clinical trials started using stem cell replacement therapy, and the induced pluripotent stem cells (iPSCs) technology combined with CRISPR-Cas9 gene editing has launched a new era in gene therapy for the treatment of neurological disorders. Here, we summarize the latest findings in this research field and discuss their clinical applications, emphasizing the relevance of recent studies in the development of innovative stem cell and gene editing therapeutic approaches. Even though tumorigenicity and immunogenicity are existing hurdles, we report how recent progress has tackled them, making engineered stem cell transplantation therapy a realistic option.
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Disruption of diphthamide synthesis genes and resulting toxin resistance as a robust technology for quantifying and optimizing CRISPR/Cas9-mediated gene editing.
We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation. PM resistance indicates integration. The robustness and generalizability of the method were demonstrated by quantifying the frequency of gene inactivation and cassette integration under different editing approaches: CRISPR/Cas9-mediated complete inactivation was ~30-50-fold more frequent than cassette integration. Mono-allelic inactivation without integration occurred >100-fold more frequently than integration. Assessment of gRNA length confirmed 20mers to be most effective length for inactivation, while 16-18mers provided the highest overall integration efficacy. The overall efficacy was ~2-fold higher for CRISPR/Cas9 than for zinc-finger nuclease and was significantly increased upon modulation of non-homologous end joining or homology-directed repair. The frequencies and ratios of editing events were similar for two different DPH genes (independent of the target sequence or chromosomal location), which indicates that the optimization parameters identified with this method can be generalized.
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Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration.
BACKGROUND: The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR-Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency. METHOD: Here we report the development of a multicolour fluorescence assay for studying CRISPR-Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR-Cas9 strategies. RESULT: We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration. CONCLUSION: Our results highlight the need for a more stringent assessment of CRISPR-Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.
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