Genome Editing Methods Research Articles

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates.

CRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method with no requirement of donor templates for convenient and efficient gene knockout in Xanthomonas oryzae pv. oryzae (Xoo), one of the most important bacterial pathogens on rice, by employing the heterologous CRISPR/Cas12a from Francisella novicida and NHEJ proteins from Mycobacterium tuberculosis. FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled multiplex genome editing, gene cluster deletion, and plasmid curing in the Xoo PXO99A strain. Accordingly, a non-TAL effector-free polymutant strain PXO99AD25E, which lacks all 25 xop genes involved in Xoo pathogenesis, has been engineered through iterative genome editing. Whole-genome sequencing analysis indicated that FnCas12a did not have a noticeable off-target effect. In addition, we revealed that these strategies are also suitable for targeted genome editing in another bacterial plant pathogen Pseudomonas syringae pv. tomato (Pst). We believe that our bacterial genome editing method will greatly expand the CRISPR study on microorganisms and advance our understanding of the physiology and pathogenesis of Xoo.

Enhanced production of d-pantothenic acid in Corynebacterium glutamicum using an efficient CRISPR–Cpf1 genome editing method

BackgroundCorynebacterium glutamicum has industrial track records for producing a variety of valuable products such as amino acids. Although CRISPR-based genome editing technologies have undergone immense developments in recent years, the suicide-plasmid-based approaches are still predominant for C. glutamicum genome manipulation. It is crucial to develop a simple and efficient CRISPR genome editing method for C. glutamicum.ResultsIn this study, we developed a RecombinAtion Prior to Induced Double-strand-break (RAPID) genome editing technology for C. glutamicum, as Cpf1 cleavage was found to disrupt RecET-mediated homologous recombination (HR) of the donor template into the genome. The RAPID toolbox enabled highly efficient gene deletion and insertion, and notably, a linear DNA template was sufficient for gene deletion. Due to the simplified procedure and iterative operation ability, this methodology could be widely applied in C. glutamicum genetic manipulations. As a proof of concept, a high-yield D-pantothenic acid (vitamin B5)-producing strain was constructed, which, to the best of our knowledge, achieved the highest reported titer of 18.62 g/L from glucose only.ConclusionsWe developed a RecET-assisted CRISPR–Cpf1 genome editing technology for C. glutamicum that harnessed CRISPR-induced DSBs as a counterselection. This method is of great importance to C. glutamicum genome editing in terms of its practical applications, which also guides the development of CRISPR genome editing tools for other microorganisms.

Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia.

Genome editing toolboxes are essential for the exploration and exploitation of non-conventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The non-conventional yeast Candida intermedia is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for C. intermedia, based on electroporation and gene deletion cassettes containing the Candida albicans NAT1 dominant selection marker flanked by 1,000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the ADE2 gene originally resulted in <1% targeting efficiencies, suggesting that C. intermedia mainly uses non-homologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split marker technique proved to be a quick and reliable method for generating gene deletions in C. intermedia, which opens the possibility to uncover and enhance its cell factory potential.

Mitochondria in Huntington's disease: implications in pathogenesis and mitochondrial-targeted therapeutic strategies.

Huntington's disease is a genetic disease caused by expanded CAG repeats on exon 1 of the huntingtin gene located on chromosome 4. Compelling evidence implicates impaired mitochondrial energetics, altered mitochondrial biogenesis and quality control, disturbed mitochondrial trafficking, oxidative stress and mitochondrial calcium dyshomeostasis in the pathogenesis of the disorder. Unfortunately, conventional mitochondrial-targeted molecules, such as cysteamine, creatine, coenzyme Q10, or triheptanoin, yielded negative or inconclusive results. However, future therapeutic strategies, aiming to restore mitochondrial biogenesis, improving the fission/fusion balance, and improving mitochondrial trafficking, could prove useful tools in improving the phenotype of Huntington's disease and, used in combination with genome-editing methods, could lead to a cure for the disease.

Editing the Genome of the Golden Hamster (Mesocricetus auratus).

The golden (Syrian) hamster (Mesocricetus auratus) is a small rodent belonging to the Cricetidae family. Golden hamsters have several unique characteristics that are advantageous in the study of reproductive and developmental biology: a highly stable 4-day estrous cycle, a high responsiveness to conventional superovulation methods, and a shortest gestation period (16days) known among eutherian mammals. Besides these advantages, the technical ease of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in this species has contributed much to our understanding of the basic mechanisms of mammalian fertilization. However, the exceptionally strong in vitro developmental block of hamster embryos, especially at the two-cell stage, has hampered the production of genetically modified hamsters, which has resulted in limited use of this species for biomedical research. However, the recently developed in vivo genome editing method (improved genome editing via oviductal nucleic acid delivery, i-GONAD) has overcome this shortcoming and made production of gene-edited hamsters much easier than before. This method has the potential to provide a means of reexamining genes whose functions cannot be identified using mouse models, thus leading to the better understanding of gene functions in mammals. In this chapter, we present our procedure for editing the genome of the golden hamster using i-GONAD.

Nonviral Ex Vivo Genome Editing in Mouse Bona Fide Hematopoietic Stem Cells with CRISPR/Cas9.

Knock-in therapy, in which an insertion site can be controlled, would be more suitable for the treatment of genetic blood disorders as compared to conventional gene therapy with lentivirus vectors that introduce genes into the genome randomly. Recent advancements in genome editing technology have substantially improved the knock-in efficiency, making it a reality. We present the details of a virus-free CRISPR/Cas9-based genome editing method for bona fide mouse hematopoietic stem cells.

Can gene expression delay aging and increase longevity?

The reality of interacting genes and proteins is vastly complex and intricate with many unknowns which currently prevent us from solving the anti-ageing equation in terms of gene expression. Molecular studies involving genome modification should take into consideration the importance of apparently opposing genes and proteins which when seen as a whole, can orchestrate an optimal systemic balance. Gene expression is often a double-edged sword with positive effects turning negative when a particular gene is overexpressed. For this reason, the US National Academy of Sciences and the National Academy of Medicine have placed stringent restrictions on the promising genome editing methods to be used only for the specific DNA sequence associated with a transmissible genetic disease. Rejuvenation as a result of gene expression is still an open area of research with studies that have either been limited to in vitro research or clinical studies claiming success based on a limited perspective that selectively emphasises the benefits of certain genes while leaving the negative outcomes in the shadows of their silence. For example, some investigators relate the presence of ZMPSTE24, IGF1R, NGF4, EEF2, EIF4FBP1 CCL18, and other genes with “rejuvenation,” selectively focusing on potential benefits while ignoring the involvement of some of these genes in malignancies and inflammation. Well-controlled gene expression molecular experimental studies with mechanotherapy and effortless exercise have limited themselves to the observation of increased slow skeletal genes associated with muscle growth that do not present any adverse side effects. Overall, exercise and nutrition are still the safest and most ethical methods of gene expression at least until genome editing can be extended to delaying ageing, in the near or distant future. As observed in the research analysing gene expression as the result of different types of exercise, inflammatory events are counterbalanced by antagonizing anti-inflammatory ones. This signifies optimal biological homeostasis because health depends on the harmonious interaction of opposite processes levelling and stabilizing each other. Exercise modalities and lifestyle are still representing the cornerstone of delaying ageing and the most riskless method of increasing longevity by triggering processes that balance each other.

An efficient marker-free genome editing method for Aspergillus niger

Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A. niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A. niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A. niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A. niger.

Recent advances in crop transformation technologies.

Agriculture is experiencing a technological inflection point in its history, while also facing unprecedented challenges posed by human population growth and global climate changes. Key advancements in precise genome editing and new methods for rapid generation of bioengineered crops promise to both revolutionize the speed and breadth of breeding programmes and increase our ability to feed and sustain human population growth. Although genome editing enables targeted and specific modifications of DNA sequences, several existing barriers prevent the widespread adoption of editing technologies for basic and applied research in established and emerging crop species. Inefficient methods for the transformation and regeneration of recalcitrant species and the genotype dependency of the transformation process remain major hurdles. These limitations are frequent in monocotyledonous crops, which alone provide most of the calories consumed by human populations. Somatic embryogenesis and de novo induction of meristems - pluripotent groups of stem cells responsible for plant developmental plasticity - are essential strategies to quickly generate transformed plants. Here we review recent discoveries that are rapidly advancing nuclear transformation technologies and promise to overcome the obstacles that have so far impeded the widespread adoption of genome editing in crop species.

A Decade of CRISPR-Cas Gnome Editing in C. elegans.

CRISPR-Cas allows us to introduce desired genome editing, including mutations, epitopes, and deletions, with unprecedented efficiency. The development of CRISPR-Cas has progressed to such an extent that it is now applicable in various fields, with the help of model organisms. C. elegans is one of the pioneering animals in which numerous CRISPR-Cas strategies have been rapidly established over the past decade. Ironically, the emergence of numerous methods makes the choice of the correct method difficult. Choosing an appropriate selection or screening approach is the first step in planning a genome modification. This report summarizes the key features and applications of CRISPR-Cas methods using C. elegans, illustrating key strategies. Our overview of significant advances in CRISPR-Cas will help readers understand the current advances in genome editing and navigate various methods of CRISPR-Cas genome editing.

Is CRISPR/Cas9 a way forward to fast-track genetic improvement in commercial palms? Prospects and limits.

Commercially important palms (oil palm, coconut, and date palm) are widely grown perennial trees with tremendous commercial significance due to food, edible oil, and industrial applications. The mounting pressure on the human population further reinforces palms' importance, as they are essential crops to meet vegetable oil needs around the globe. Various conventional breeding methods are used for the genetic improvement of palms. However, adopting new technologies is crucial to accelerate breeding and satisfy the expanding population's demands. CRISPR/Cas9 is an efficient genome editing tool that can incorporate desired traits into the existing DNA of the plant without losing common traits. Recent progress in genome editing in oil palm, coconut and date palm are preliminarily introduced to potential readers. Furthermore, detailed information on available CRISPR-based genome editing and genetic transformation methods are summarized for researchers. We shed light on the possibilities of genome editing in palm crops, especially on the modification of fatty acid biosynthesis in oil palm. Moreover, the limitations in genome editing, including inadequate target gene screening due to genome complexities and low efficiency of genetic transformation, are also highlighted. The prospects of CRISPR/Cas9-based gene editing in commercial palms to improve sustainable production are also addressed in this review paper.

Prospective Advances in Genome Editing Investigation.

Genomic editing technology has been developed since 2010 through the use of some techniques, such as the clustered regularly interspaced short palindromic repeat (CRISPR) DNA sequences/CRISPR-associated (Cas) type-9 method, or through genetic manipulation tools derived from host response systems from some microbes (e.g., bacteria) against plasmids and viruses. The introduction of the CRISPR/Cas9 method as a genome-editing instrument represented an important step in the advancement of the genome-editing method thanks to the ease and effectiveness of use as well as the great adaptability to different biomedical areas. This paragraph will discuss all conceived technologies and new perspectives that can be applied in treating some associated genetic disorders, such as cardiovascular diseases, metabolic diseases, inflammatory diseases, and tumors by means of reversible and modulating control of gene expression epigenetics using genetic editing techniques.

Abstract 10925: A Small Molecule Inhibitor of Sarcomere Contractility Acutely Relieves Prolonged Relaxation in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes With Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases primarily caused by mutations in sarcomeric genes. However, HCM is also associated with mutations in non-sarcomeric proteins and a Finnish founder mutation for HCM in non-sarcomeric protein junctophilin-2 (JPH2) has been identified. MYK-461 is a recently described mechanistically novel small molecule that acts at the sarcomere to specifically inhibit contractility that has been proposed as a treatment for HCM. Here, we use MYK-461 to test whether direct reduction in contractility is sufficient to relieve phenotypic characteristics in human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) carrying JPH2 mutation T161K. Hypothesis: Currently all HCM patients are treated the same regardless of the underlying genetic defect. Current medication does not prevent or stop the thickening of the heart muscle; only the symptoms and arrhythmias can be controlled with beta-blockers or calcium blockers. MYK-461 decreases adenosine triphosphatase activity of the cardiac myosin heavy chain to reduce contractility, thus modifying the characteristic pathology of HCM. Methods: Skin fibroblasts from a Finnish patient with HCM causing JPH2 p.(Thr161Lys) were reprogrammed into hiPSCs and further differentiated into CMs. As a control line, an isogenic counterpart was generated using the CRISPR/Cas9 genome editing method. We evaluated the effect of MYK-461 treatment to T161K-hiPSC-CMs by their functional characteristics associated with JPH2 mutation. Results: By video microscopy, we identified increased contractility (BPM; 41±6 SEM, n=9) and prolonged diastolic phase (ms; 394.8±29.7 SEM, n=9) in T161K-hiPSC-CMs. Treatment with MYK-461 reduced contractility (BPM; 36±3 SEM, n=10) and relaxation phase (ms; 293.1±33.6 SEM, n=10) in a dose-dependent manner, displaying functional characteristics closer to isogenic-hiPSC-CMs control group. Conclusions: In conclusion, the results demonstrate that acute reduction in contractility is sufficient to relieve prolonged relaxation. Further, these studies suggest that T161K-hiPSC-CMs provide adequate in vitro platform for modelling the functional effect of MYK-461.

Caenorhabditis elegans as a Model for the Effects of Phytochemicals on Mitochondria and Aging.

The study of aging is an important topic in contemporary research. Considering the demographic changes and the resulting shifts towards an older population, it is of great interest to preserve youthful physiology in old age. For this endeavor, it is necessary to choose an appropriate model. One such model is the nematode Caenorhabditis elegans (C. elegans), which has a long tradition in aging research. In this review article, we explore the advantages of using the nematode model in aging research, focusing on bioenergetics and the study of secondary plant metabolites that have interesting implications during this process. In the first section, we review the situation of aging research today. Conventional theories and hypotheses about the ongoing aging process will be presented and briefly explained. The second section focuses on the nematode C. elegans and its utility in aging and nutrition research. Two useful genome editing methods for monitoring genetic interactions (RNAi and CRISPR/Cas9) are presented. Due to the mitochondria's influence on aging, we also introduce the possibility of observing bioenergetics and respiratory phenomena in C. elegans. We then report on mitochondrial conservation between vertebrates and invertebrates. Here, we explain why the nematode is a suitable model for the study of mitochondrial aging. In the fourth section, we focus on phytochemicals and their applications in contemporary nutritional science, with an emphasis on aging research. As an emerging field of science, we conclude this review in the fifth section with several studies focusing on mitochondrial research and the effects of phytochemicals such as polyphenols. In summary, the nematode C. elegans is a suitable model for aging research that incorporates the mitochondrial theory of aging. Its living conditions in the laboratory are optimal for feeding studies, thus enabling bioenergetics to be observed during the aging process.

ACtivE: Assembly and CRISPR-Targeted in Vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time.

Thanks to its sophistication, the CRISPR/Cas system has been a widely used yeast genome editing method. However, CRISPR methods generally rely on preassembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose an alternative method, Assembly and CRISPR-targeted in vivo Editing (ACtivE), that only relies on in vivo assembly of linear DNA fragments for plasmid and donor DNA construction. Thus, depending on the user's need, these parts can be easily selected and combined from a repository, serving as a toolkit for rapid genome editing without any expensive reagent. The toolkit contains verified linear DNA fragments, which are easy to store, share, and transport at room temperature, drastically reducing expensive shipping costs and assembly time. After optimizing this technique, eight loci proximal to autonomously replicating sequences (ARS) in the yeast genome were also characterized in terms of integration and gene expression efficiencies and the impacts of the disruptions of these regions on cell fitness. The flexibility and multiplexing capacity of the ACtivE were shown by constructing a β-carotene pathway. In only a few days, >80% integration efficiency for single gene integration and >50% integration efficiency for triplex integration were achieved on Saccharomyces cerevisiae BY4741 from scratch without using in vitro DNA assembly methods, restriction enzymes, or extra cloning steps. This study presents a standardizable method to be readily employed to accelerate yeast genome engineering and provides well-defined genomic location alternatives for yeast synthetic biology and metabolic engineering purposes.

Immunohistochemical Staining of the Mosquito Ear.

Immunohistochemistry has played a major role in improving our understanding of the anatomy and function of the nervous system. The use of fluorescent dyes that label different antigens reveals how biological tissues are built and how interactions between cells take place. Obtaining this information is particularly important in the case of the mosquito ear given its highly complex anatomy. This protocol describes an immunohistochemical technique to stain the mosquito ear. The first steps of the procedure include the embedding of the tissue in albumin-gelatin and its sectioning into thin slices to allow antibody penetration. The immunohistochemical procedure can be exploited to detect protein expression and localization by using antibodies specifically raised against the protein of interest or that recognize epitope tags fused to proteins using genome editing methods.

Current updates of CRISPR/Cas9-mediated genome editing and targeting within tumor cells: an innovative strategy of cancer management.

Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9), an adaptive microbial immune system, has been exploited as a robust, accurate, efficient and programmable method for genome targeting and editing. This innovative and revolutionary technique can play a significant role in animal modeling, in vivo genome therapy, engineered cell therapy, cancer diagnosis and treatment. The CRISPR/Cas9 endonuclease system targets a specific genomic locus by single guide RNA (sgRNA), forming a heteroduplex with target DNA. The Streptococcus pyogenes Cas9/sgRNA:DNA complex reveals a bilobed architecture with target recognition and nuclease lobes. CRISPR/Cas9 assembly can be hijacked, and its nanoformulation can be engineered as a delivery system for different clinical utilizations. However, the efficient and safe delivery of the CRISPR/Cas9 system to target tissues and cancer cells is very challenging, limiting its clinical utilization. Viral delivery strategies of this system may have many advantages, but disadvantages such as immune system stimulation, tumor promotion risk and small insertion size outweigh these advantages. Thus, there is a desperate need to develop an efficient non-viral physical delivery system based on simple nanoformulations. The delivery strategies of CRISPR/Cas9 by a nanoparticle-based system have shown tremendous potential, such as easy and large-scale production, combination therapy, large insertion size and efficient in vivo applications. This review aims to provide in-depth updates on Streptococcus pyogenic CRISPR/Cas9 structure and its mechanistic understanding. In addition, the advances in its nanoformulation-based delivery systems, including lipid-based, polymeric structures and rigid NPs coupled to special ligands such as aptamers, TAT peptides and cell-penetrating peptides, are discussed. Furthermore, the clinical applications in different cancers, clinical trials and future prospects of CRISPR/Cas9 delivery and genome targeting are also discussed.

Perspectives on Genetic Medicine for Cystic Fibrosis.

Like any inherited protein deficiency disease, cystic fibrosis (CF) is a good candidate for gene replacement therapy. Despite the tremendous efforts of scientists worldwide invested in developing this approach, it did not lead to the expected results for various reasons discussed in this review. At the same time, the emergence of new methods of genome editing, as well as their latest modifications, makes it possible to bypass some of the problems of "classical" CF gene therapy. The review examines potential therapeutic agents for CF gene therapy, methods and routes of delivery, as well as discusses the problem of target cells for defect correction. Based on the results of these studies, editing genetic defects in the basal cells of the lungs and their counterparts in other organs will make it possible to create a drug for treating CF with a single administration.

A Split CRISPR-Cpf1 Platform for Inducible Gene Activation.

The CRISPR-Cpf1 also known as Cas12a is an RNA-guided endonuclease similar to CRISPR-Cas9. Combining the CRISPR-Cpf1 with optogenetics technology, we have engineered photoactivatable Cpf1 (paCpf1) to precisely control the genome sequence in a spatiotemporal manner. We also identified spontaneously activated split Cpf1 and thereby developed a potent dCpf1 split activator, which has the potential to activate endogenous target genes. Here we describe a method for optogenetic endogenous genome editing using paCpf1 in mammalian cells. Furthermore, we show a method for endogenous gene activation using dCpf1 split activator in mammalian cells and mice.

Current advances and future trend of nanotechnology as microalgae-based biosensor

Biosensors are devices that convert biological responses into electrical signals. Biosensors can be utilised in a variety of applications, including environmental and water quality monitoring, as well as medical applications. Thus, researchers have been working on them and experimenting with new nanofabrication techniques. Nanomaterials like graphene, carbon nanotubes, and nanospheres have provided new and appealing platforms for developing biosensors with improved sensitivity and performance due to their unique features. Microalgae have been used widely in biosensing applications as bioreceptor because they are less likely to be affected by physiochemical changes, in addition to their uses in medical, environmental, industrial, and commercial applications. In this review, we highlight the recent immobilization techniques, support materials like nanomaterials for microalgae immobilization, microfluidics and lab on a chip, the latest genome editing methods, and microalgae applications in medical, environmental, food, and agricultural sectors.