May I Cut in? Gene Editing Approaches in Human Induced Pluripotent Stem Cells.

In last two decades, gene editing technology has been developed rapidly, and gene editing tools have become more and more efficient. The most widely used tools are DNA nucleases, like zinc-finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (CRISPR/Cas). Especially the CRISPR/Cas9 technology and its application, which has been awarded Ten Scientific Breakthroughs of the Year by Nature for three consecutive years since its publication, have set off an unprecedented upsurge in gene editing research. The birth of Gene Editing Infant has triggered medical ethical issues, which further makes gene editing technology the focus of public discussion and attention. CRISPR/Cas9 technology has been widely using in biomedical field attributed to its easy operation, high efficiency and low cost. In this paper, the research progress and application of CRISPR/Cas9 gene editing technology in liver diseases will be reviewed. Key words: Gene editing; Clustered regularly interspaced short palindromic repeats/Cas9; Liver diseases; Hepatic injury and repair

Background:Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods.Materials and Methods:At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F’ that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry.Results:The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) (P < 0.0001).Conclusion:According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.

This review will highlight some of the recent advances in genome engineering with applications for both clinical and basic science investigations of HIV-1. Over the last year, the field of HIV cure research has seen major breakthroughs with the success of the first phase I clinical trial involving gene editing of CCR5 in patient-derived CD4(+) T cells. This first human use of gene-editing technology was accomplished using zinc finger nucleases (ZFNs). Zinc finger nucleases and the advent of additional tools for genome engineering, including transcription activator-like effector nucleases (TALENS) and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, have made gene editing remarkably simple and affordable. Here we will discuss the different gene-editing technologies, the use of gene editing in HIV research over the past year, and potential applications of gene editing for both in-vitro and in-vivo studies. Genome-engineering technologies have rapidly progressed over the past few years such that these systems can be easily applied in any laboratory for a variety of purposes. For HIV-1, upcoming clinical trials will determine if gene editing can provide the long-awaited functional cure. In addition, manipulation of host genomes, whether in vivo or in vitro, can facilitate development of better animal models and culture methods for studying HIV-1 transmission, pathogenesis, and virus-host interactions.

Versatile and multifaceted CRISPR/Cas gene editing tool for plant research

At present, the main gene editing tools encompass TREN, Zinc Finger Nucleases (ZFN), clustered regularly interspaced short palindromic repeats (CRISPR), and Transcription Activator-Like Effector Nucleases (TALEN). In this study, we introduce an overview of the three gene editing methodologies and discuss their current clinical applications. In addition, we suggest some trends and future applications within the field of gene editing. ZFNs represent one of the pioneering technologies, demonstrating significant efficacy in mitigating a multitude of genetic diseases and finding applications in agriculture. Yet, this technology contains intricate processes and produces substantial costs when implemented. TALENs have already been employed across various domains. In the medical field, they have been successfully applied in the treatment of leukemia in infants. However, TALENs are being replaced by CRISPR due to the superior efficiency of CRISPR. CRISPR, consisting of six components, exhibits considerable promise in the medical realm, particularly in the context of treating diseases such as Alzheimer's disease (AD). In the realm of genetic engineering, it can collaborate with B cells to rectify specific genes within the human genome, which have been tested in experiments. In the future, it can be used in many fields, including agriculture and nucleic acid testing.

Christmas disease, until now, has been considered incurable -a disease for life.

Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency

Antiretroviral therapy has prolonged the lives of people living with human immunodeficiency virus type 1 (HIV-1), transforming the disease into one that can be controlled with lifelong therapy. The search for an HIV-1 vaccine has plagued researchers for more than three decades with little to no success from clinical trials. Due to these failures, scientists have turned to alternative methods to develop next generation therapeutics that could allow patients to live with HIV-1 without the need for daily medication. One method that has been proposed has involved the use of a number of powerful gene editing tools; Zinc Finger Nucleases (ZFN), Transcription Activator–like effector nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 to edit the co-receptors (CCR5 or CXCR4) required for HIV-1 to infect susceptible target cells efficiently. Initial safety studies in patients have shown that editing the CCR5 locus is safe. More in depth in vitro studies have shown that editing the CCR5 locus was able to inhibit infection from CCR5-utilizing virus, but CXCR4-utilizing virus was still able to infect cells. Additional research efforts were then aimed at editing the CXCR4 locus, but this came with other safety concerns. However, in vitro studies have since confirmed that CXCR4 can be edited without killing cells and can confer resistance to CXCR4-utilizing HIV-1. Utilizing these powerful new gene editing technologies in concert could confer cellular resistance to HIV-1. While the CD4, CCR5, CXCR4 axis for cell-free infection has been the most studied, there are a plethora of reports suggesting that the cell-to-cell transmission of HIV-1 is significantly more efficient. These reports also indicated that while broadly neutralizing antibodies are well suited with respect to blocking cell-free infection, cell-to-cell transmission remains refractile to this approach. In addition to stopping cell-free infection, gene editing of the HIV-1 co-receptors could block cell-to-cell transmission. This review aims to summarize what has been shown with regard to editing the co-receptors needed for HIV-1 entry and how they could impact the future of HIV-1 therapeutic and prevention strategies.

Microalgae, a kind of unicellular photoautotrophs that widely exist in marine and freshwater. Microalgae can accumulate a great deal of metabolites in the process of growth, then they have high-value in bioenergy, food production, health care and animal feed. Although microalgae has great potential of development, its practical application is still slow because of its various biological species, incomplete genetic information research, complex industrial production process and so on. In recent years, due to the rapid development of genomics research, especially the emergence of new gene editing techniques, people can quickly efficiently and fully figure out the genetic and molecular information of microalgae. Therefore, wide attention has been paid to the application of new gene editing technology in microalgae breeding and microalgae fermentation engineering. Based on this, this paper briefly introduces some new gene editing techniques, such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats, CRISPR-associated system, and summarizes the application progress of the above techniques in microalgae gene editing and engineering.

Gene editing offers dietary benefits

Acquired immunodeficiency syndrome (AIDS) is a deadly immunological disease that affects the immune system and becomes more common in recent years, which is brought on by the inflection of human immunodeficiency (HIV). The treatment of AIDS has always been a major challenge for the medical community to overcome because the existing treatment methods are not universal. At present, the scientific community is focusing on gene editing therapy, and the feasible tools are zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9). Since gene editing technology has advanced so quickly, gene editing therapy is now thought to be one of the most promising AIDS treatment. Though these systems are powerful and efficient, there are still several serious challenges that scientists should face. The clinical safety issues have always existed and been controversial, especially after He Jiankui’s case that editing AIDS-resistance human embryos coming to the light. And the legal regulations were strongly criticized due to gaps in the bottom line of scientific research. This review summarizes existing applications of gene editing in AIDS with the safety and ethical issues of clinical application, searching for the relevant solutions to the current challenges.

CRISPR Genome Editing: Into the Second Decade

Recent advances in high throughout DNA sequencing technologies have revolutionized for better understanding of structure-functional relationships of genes in identifying trait-associated transcriptomes and their regulated gene expressions. Subsequent breakthroughs in gene editing technologies such as zinc finger nucleases, transcriptional activator-like effect or nucleases (TALEN) and CRISPR (clustered regularly interspaced short palindromic repeats) determined chromosomal loci so as to understand gene functions in vivo. Such editing technologies are now being implemented in many laboratories due to an affordable cost and easiness of techniques. Targeted gene delivery and disruptions are now not only restricted to standard cell lines or stem cells, but also primary cell lines and nonmodel agriculturally important species. Progress and implications of gene integration and disruptions in food fishes like salmon, carps, etc. will be highlighted. The positive impacts on myostatin gene (negative growth hormone regulator) disruption mediated muscular growth have been documented. Transposon mediated gene integration technologies for value-additions to small indigenous aquarium fishes by expressing attractive fluorescent color genes could be the future of rainbow revolution. Issues linked with further-tuning with regards to improved efficacy and specificity, while reducing offtarget effects of gene editing tools will be addressed. There are health and environmental concerns with genetically modified organisms (GMOs). CRISPR/Cas9 mediated editing generates indels and hence supposed to be free from transgene-nontoxic and non-allergen. Scientific progress regarding to generate genetically modified carps; those could well be cultivated in a confinement and at the same time economically profitable; will be highlighted. Emphasis should be given for transfer these technologies from the laboratory to land for the development of a consumer-friendly sustainable farming system.

ABSTRACTIn the past few years, new technologies have arisen that enable higher efficiency of gene editing. With the increase ease of using gene editing technologies, it is important to consider the best method for transferring new genetic material to livestock animals. Microinjection is a technique that has proven to be effective in mice but is less efficient in large livestock animals. Over the years, a variety of methods have been used for cloning as well as gene transfer including; nuclear transfer, sperm mediated gene transfer (SMGT), and liposome-mediated DNA transfer. This review looks at the different success rate of these methods and how they have evolved to become more efficient. As well as gene editing technologies, including Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the most recent clustered regulatory interspaced short palindromic repeats (CRISPRs). Through the advancements in gene-editing technologies, generating transgenic animals is now more accessible and affordable. The goals of producing transgenic animals are to 1) increase our understanding of biology and biomedical science; 2) increase our ability to produce more efficient animals; and 3) produce disease resistant animals. ZFNs, TALENs, and CRISPRs combined with gene transfer methods increase the possibility of achieving these goals.

Genome editing is a relevant, versatile, and preferred tool for crop improvement, as well as for functional genomics. In this review, we summarize the advances in gene-editing techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated with the Cas9 and Cpf1 proteins. These tools support great opportunities for the future development of plant science and rapid remodeling of crops. Furthermore, we discuss the brief history of each tool and provide their comparison and different applications. Among the various genome-editing tools, CRISPR has become the most popular; hence, it is discussed in the greatest detail. CRISPR has helped clarify the genomic structure and its role in plants: For example, the transcriptional control of Cas9 and Cpf1, genetic locus monitoring, the mechanism and control of promoter activity, and the alteration and detection of epigenetic behavior between single-nucleotide polymorphisms (SNPs) investigated based on genetic traits and related genome-wide studies. The present review describes how CRISPR/Cas9 systems can play a valuable role in the characterization of the genomic rearrangement and plant gene functions, as well as the improvement of the important traits of field crops with the greatest precision. In addition, the speed editing strategy of gene-family members was introduced to accelerate the applications of gene-editing systems to crop improvement. For this, the CRISPR technology has a valuable advantage that particularly holds the scientist’s mind, as it allows genome editing in multiple biological systems.