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Implementing CRISPR-Cas9 Yeast Practicals into Biology Curricula.
CRISPR-Cas9 is a genome-editing technique that has been widely adopted thanks to its simplicity, efficiency, and broad application potential. Due to its advantages and pervasive use, there have been attempts to include this method in the existing curricula for students majoring in various disciplines of biology. In this perspective, we summarize the existing CRISPR-Cas courses that harness a well-established model organism: baker's yeast, Saccharomyces cerevisiae. As an example, we present a detailed description of a fully hands-on, flexible, robust, and cost-efficient practical CRISPR-Cas9 course, where students participate in yeast genome editing at every stage-from the bioinformatic design of single-guide RNA, through molecular cloning and yeast transformation, to the final confirmation of the introduced mutation. Finally, we emphasize that in addition to providing experimental skills and theoretical knowledge, the practical courses on CRISPR-Cas represent ideal platforms for discussing the ethical implications of the democratization of biology.
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Improvement of the CRISPR-Cas9 mediated gene disruption and large DNA fragment deletion based on a chimeric promoter in Acremonium chrysogenum.
Acremonium chrysogenum has been employed in the industrial production of cephalosporin C (CPC). However, there are still some impediments to understanding the regulation of CPC biosynthesis and improving strains due to the difficulty of genetic manipulation in A. chrysogenum, especially in the CPC high-producing strain C10. Here, an improved CRISPR-Cas9 system was constructed based on an U6/tRNA chimeric promoter. Using this system, high efficiency for single gene disruption was achieved in C10. In addition, double loci were simultaneously targeted when supplying with the homology-directed repair templates (donor DNAs). Based on this system, large DNA fragments up to 31.5 kb for the yellow compound sorbicillinoid biosynthesis were successfully deleted with high efficiency. Furthermore, CPC production was significantly enhanced when the sorbicillinoid biosynthetic genes were knocked out. This study provides a powerful tool for gene editing and strain improvement in A. chrysogenum.
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Genome Editing and Hematologic Malignancy.
The modern genomic era has seen remarkable advancement in our understanding of the molecular basis for disease, yet translation of basic discoveries into new disease treatments has arguably lagged behind. Recently, breakthroughs in genome editing technologies have created hope for their potential to directly treat the genetic causes of disease. Like any therapeutic intervention, genome editing should be considered in light of its potential risks and benefits. In this review, we highlight the promise of genome editing therapies, as well as the conceptual and technical barriers to their clinical application, with a special emphasis on hematologic malignancies.
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Activation of homology-directed DNA repair plays key role in CRISPR-mediated genome correction.
Gene editing for the cure of inborn errors of metabolism (IEMs) has been limited by inefficiency of adult hepatocyte targeting. Here, we demonstrate that in utero CRISPR/Cas9-mediated gene editing in a mouse model of hereditary tyrosinemia type 1 provides stable cure of the disease. Following this, we performed an extensive gene expression analysis to explore the inherent characteristics of fetal/neonatal hepatocytes that make them more susceptible to efficient gene editing than adult hepatocytes. We showed that fetal and neonatal livers are comprised of proliferative hepatocytes with abundant expression of genes involved in homology-directed repair (HDR) of DNA double-strand breaks (DSBs), key for efficient gene editing by CRISPR/Cas9. We demonstrated the same is true of hepatocytes after undergoing a regenerative stimulus (partial hepatectomy), where post-hepatectomy cells show a higher efficiency of HDR and correction. Specifically, we demonstrated that HDR-related genome correction is most effective in the replicative phase, or S-phase, of an actively proliferating cell. In conclusion, this study shows that taking advantage of or triggering cell proliferation, specifically DNA replication in S-phase, may serve as an important tool to improve efficiency of CRISPR/Cas9-mediated genome editing in the liver and provide a curative therapy for IEMs in both children and adults.
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Electroporation-mediated genome editing in vitrified/warmed mouse zygotes created by IVF via ultra-superovulation.
Recently, genome editing in mouse zygotes has become convenient and scalable, in association with various technological developments and improvements such as novel nuclease tools, alternative delivery methods, and contemporary reproductive engineering techniques. We have so far demonstrated the applicability of ultra-superovulation, in vitro fertilization (IVF), and vitrification/warming of zygotes in microinjection-mediated mouse genome editing. Moreover, an electroporation-mediated method has rapidly become established for simple gene knockout and small precise modifications including single amino acid substitutions. Here, we present an updated example of an application coupling the following three latest technologies: 1) CRISPR-Cas9 ribonucleoprotein as the most convenient genome-editing reagent, 2) electroporation as the most effortless delivery method, and 3) cryopreserved oocytes created by IVF via ultra-superovulation as the most animal welfare- and user-friendly strategy. We successfully created gene knockout and knock-in mice carrying insertion/deletion mutations and single amino acid substitutions, respectively, using the streamlined production system of mouse genome editing described above, referred to as the CREATRE (CARD-based Reproductive Engineering-Assisted Technology for RNP Electroporation) system. Owing to its accessibility, robustness, and high efficiency, we believe that our CREATRE protocol will become widely used globally for the production of genome-edited mice.
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Employing CRISPR-Cas9 to Enhance T Cell Effector Function.
As part of the adaptive immune system, T cells are critical to maintain immune homeostasis. T cells provide protective immunity by killing infected cells and combatting cancerous cells. To do so, T cells produce and secrete effector molecules, such as granzymes, perforin, and cytokines such as tumor necrosis factor α and interferon γ. However, in immune suppressive environments, such as tumors, T cells gradually lose the capacity to perform their effector function. One way T cell effector function can be enhanced is through genetic engineering with tools such as clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9). This protocol explains in a step-by-step fashion how to perform a controlled electroporation-based CRISPR experiment to enhance human T cell effector function. Of note, these steps are suitable for CRISPR-mediated genome editing in T cells in general and can thus also be used to study proteins of interest that do not influence T cell effector function.
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CRISPR/Cas9 for development of disease resistance in plants: recent progress, limitations and future prospects.
Several plant pathogens severely affect crop yield and quality, thereby threatening global food security. In order to cope with this challenge, genetic improvement of plant disease resistance is required for sustainable agricultural production, for which conventional breeding is unlikely to do enough. Luckily, genome editing systems that particularly clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has revolutionized crop improvement by enabling robust and precise targeted genome modifications. It paves the way towards new methods for genetic improvement of plant disease resistance and accelerates resistance breeding. In this review, the challenges, limitations and prospects for conventional breeding and the applications of CRISPR/Cas9 system for the development of transgene-free disease-resistant crops are discussed.
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Genome editing in Bombyx mori: New opportunities for silkworm functional genomics and the sericulture industry.
In recent years, research in life sciences has been remarkably revolutionized owing to the establishment, development and application of genome editing technologies. Genome editing has not only accelerated fundamental research but has also shown promising applications in agricultural breeding and therapy. In particular, the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology has become an indispensable tool in molecular biology owing to its high efficacy and simplicity. Genome editing tools have also been established in silkworm (Bombyx mori), a model organism of Lepidoptera insects with high economic importance. This has remarkably improved the level and scope of silkworm research and could reveal new mechanisms or targets in basic entomology and pest management studies. In this review, we summarize the progress and potential of genome editing in silkworm and its applications in functional genomic studies for generating novel genetic materials.
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Genome Editing in Staphylococcus aureus by Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection.
Methods for the genetic manipulation of S. aureus have historically proven challenging, which has hindered experimental studies of this organism. We recently developed a system for recombineering and CRISPR/Cas9-mediated counterselection in S. aureus which utilizes commercially synthesized synthetic DNA oligonucleotides as substrates for introducing precise genomic modifications into the organism and for performing lethal counterselection of unedited cells. These techniques make it possible to scalably and inexpensively engineer desired genomic changes into laboratory or clinical S. aureus strains, using electroporation to introduce the effector plasmid vectors and oligonucleotides. Here we describe detailed protocols for performing genome editing of S. aureus in order to produce isogenic strains using this system and detail general principles which are broadly applicable across a range of organisms for which equivalent systems have been established.
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Gene Editing and Human iPSCs in Cardiovascular and Metabolic Diseases.
The incidence and the burden of cardiovascular disease (CVD), coronary heart disease (CHD), type 2 diabetes mellitus (T2DM), and the metabolic syndrome are greatly increasing in our societies. Together, they account for 31% of all deaths worldwide. This chapter focuses on the role of two revolutionary discoveries that are changing the future of medicine, induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 technology, in the study, and the cure of cardiovascular and metabolic diseases.We summarize the state-of-the-art knowledge about the possibility of editing iPSC genome for therapeutic applications without hampering their pluripotency and differentiation, using CRISPR/Cas technology, in the field of cardiovascular and metabolic diseases.
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