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High-throughput methods for genome editing: the more the better.
During the last decade, targeted genome-editing technologies, especially clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) technologies, have permitted efficient targeting of genomes, thereby modifying these genomes to offer tremendous opportunities for deciphering gene function and engineering beneficial traits in many biological systems. As a powerful genome-editing tool, the CRISPR/Cas systems, combined with the development of next-generation sequencing and many other high-throughput techniques, have thus been quickly developed into a high-throughput engineering strategy in animals and plants. Therefore, here, we review recent advances in using high-throughput genome-editing technologies in animals and plants, such as the high-throughput design of targeted guide RNA (gRNA), construction of large-scale pooled gRNA, and high-throughput genome-editing libraries, high-throughput detection of editing events, and high-throughput supervision of genome-editing products. Moreover, we outline perspectives for future applications, ranging from medication using gene therapy to crop improvement using high-throughput genome-editing technologies.
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Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy.
The growing clinical success of hematopoietic stem/progenitor cell (HSPC) gene therapy (GT) relies on the development of viral vectors as portable "Trojan horses" for safe and efficient gene transfer. The recent advent of novel technologies enabling site-specific gene editing is broadening the scope and means of GT, paving the way to more precise genetic engineering and expanding the spectrum of diseases amenable to HSPC-GT. Here, we provide an overview of state-of-the-art and prospective developments of the HSPC-GT field, highlighting how advances in biological characterization and manipulation of HSPCs will enable the design of the next generation of these transforming therapeutics.
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Genetic Manipulation of Reactive Oxygen Species (ROS) Homeostasis Utilizing CRISPR/Cas9-Based Gene Editing in Rice.
Reactive oxygen species (ROS) are now recognized as key signals in plant stress responses. Adverse environmental conditions can either promote ROS production or downregulate antioxidative enzymes, leading to the alteration of redox homeostasis and activation of ROS-linked stress signaling. To uncover their signaling mechanisms and to characterize related components, genetic modification of ROS homeostasis is a central approach. CRISPR/Cas9-based genome editing system has become a powerful tool for gene mutation in a variety of organisms, including plants. Within this chapter, we describe a method that can be applied to manipulate ROS homeostasis in rice (Oryza sativa L.) utilizing CRISPR/Cas9 technology. Step-by-step protocols including the design and construction of Cas9/sgRNA, agrobacterium-mediated transformation, and mutation characterization are described. Application of this system in editing a rice catalase gene CatC, a key antioxidative enzyme in controlling ROS homeostasis, is also presented.
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Genome engineering tools for building cellular models of disease.
With the recent development of methods for genome editing of human pluripotent stem cells, study of the genetic basis of human diseases has been rapidly advancing. Genome-edited differentiated stem cells have provided new and more accurate insights into genomic underpinnings of diseases for which there have not been adequate treatments, and moving toward clinical application of genome editing holds great promise for acceleration of therapeutic translation. Here, we review recent advances in genome-editing technologies and their application to human biology in disease modeling and beyond.
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Optimized Guide RNA Selection Improves Streptococcus pyogenes Cas9 Gene Editing of Human Hematopoietic Stem and Progenitor Cells.
Ribonucleoproteins (RNPs) are frequently applied for therapeutic gene editing as well as fundamental research because the method is fast, viral free, and shows fewest off target effects. We evaluated various parameters to genetically engineer human hematopoietic stem and progenitor cells (HSPCs) using Streptococcus pyogenes Cas9 (spCas9) RNPs, and achieve gene editing efficiencies up to 80%. We find that guide RNA (gRNA) design is critical to achieve high gene editing efficiencies. However, finding effective gRNAs for HSPCs can be challenging, while the contribution of numerous in silico models is unclear. By screening more than 120 gRNAs, our data demonstrate that in silico gRNA prediction models are ineffective. In this study, we established a time- and cost-efficient in vitro transcribed gRNA screening model in K562 cells that predicts effective gRNAs for HSPCs. RNP based screening thus outperforms in silico modeling and we report that gene editing is equally efficient in distinct CD34+ HSPC subpopulations. Furthermore, no effects on cell proliferation, differentiation, or in vitro hematopoietic lineage commitment were observed. Finally, no upregulation of p21 expression was found, suggesting unperturbed HSPC homeostasis.
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CRISPR/Cas9-mediated genome editing of Shewanella oneidensis MR-1 using a broad host-range pBBR1-based plasmid.
Here, we developed an all-in-one, broad host-range CRISPR/Cas9 vector system widely applicable to genome editing of proteobacteria. Plasmid pBBR1-Cas9 was constructed by cloning the cas9 gene from Streptococcus pyogenes into the broad host-range plasmid pBBR1MCS-2. We evaluated its applicability for frameshift mutagenesis of Shewanella oneidensis MR-1. Significant cell death was observed when MR-1 cells were transformed with a pBBR1-Cas9 derivative that expressed a single-guide RNA targeting the crp gene. However, cell death was partially prevented when a donor DNA fragment containing a modified crp sequence with a frameshift mutation was introduced using the same vector. All transformants (9 colonies) contained the expected frameshift mutation in their chromosomal crp genes. These results indicate that this vector system efficiently introduced CRISPR/Cas9-mediated double-strand DNA breaks and subsequent homology-directed repair. This work provides a simple and powerful genome-editing tool for proteobacteria that can harbor pBBR1-based plasmids.
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CRISPR-based strategies in infectious disease diagnosis and therapy.
PURPOSE: CRISPR gene-editing technology has the potential to transform the diagnosis and treatment of infectious diseases, but most clinicians are unaware of its broad applicability. Derived from an ancient microbial defence system, these so-called "molecular scissors" enable precise gene editing with a low error rate. However, CRISPR systems can also be targeted against pathogenic DNA or RNA sequences. This potential is being combined with innovative delivery systems to develop new therapeutic approaches to infectious diseases. METHODS: We searched Pubmed and Google Scholar for CRISPR-based strategies in the diagnosis and treatment of infectious diseases. Reference lists were reviewed and synthesized for narrative review. RESULTS: CRISPR-based strategies represent a novel approach to many challenging infectious diseases. CRISPR technologies can be harnessed to create rapid, low-cost diagnostic systems, as well as to identify drug-resistance genes. Therapeutic strategies, such as CRISPR systems that cleave integrated viral genomes or that target resistant bacteria, are in development. CRISPR-based therapies for emerging viruses, such as SARS-CoV-2, have also been proposed. Finally, CRISPR systems can be used to reprogram human B cells to produce neutralizing antibodies. The risks of CRISPR-based therapies include off-target and on-target modifications. Strategies to control these risks are being developed and a phase 1 clinical trials of CRISPR-based therapies for cancer and monogenic diseases are already underway. CONCLUSIONS: CRISPR systems have broad applicability in the field of infectious diseases and may offer solutions to many of the most challenging human infections.
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A Code of Ethics for Gene Drive Research.
Gene drives hold promise for use in controlling insect vectors of diseases, agricultural pests, and for conservation of ecosystems against invasive species. At the same time, this technology comes with potential risks that include unknown downstream effects on entire ecosystems as well as the accidental or nefarious spread of organisms that carry the gene drive machinery. A code of ethics can be a useful tool for all parties involved in the development and regulation of gene drives and can be used to help ensure that a balanced analysis of risks, benefits, and values is taken into consideration in the interest of society and humanity. We have developed a code of ethics for gene drive research with the hope that this code will encourage the development of an international framework that includes ethical guidance of gene drive research and is incorporated into scientific practice by gaining broad agreement and adherence.
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Applications of CRISPR/Cas9 in the Mammalian Central Nervous System.
Within the central nervous system, gene regulatory mechanisms are crucial regulators of cellular development and function, and dysregulation of these systems is commonly observed in major neuropsychiatric and neurological disorders. However, due to a lack of tools to specifically modulate the genome and epigenome in the central nervous system, many molecular and genetic mechanisms underlying cognitive function and behavior are still unknown. Although genome editing tools have been around for decades, the recent emergence of inexpensive, straightforward, and widely accessible CRISPR/Cas9 systems has led to a revolution in gene editing. The development of the catalytically dead Cas9 (dCas9) expanded this flexibility even further by acting as an anchoring system for fused effector proteins, structural scaffolds, and RNAs. Together, these advances have enabled robust, modular approaches for specific targeting and modification of the local chromatin environment at a single gene. This review highlights these advancements and how the combination of powerful modulatory tools paired with the versatility of CRISPR-Cas9-based systems offer great potential for understanding the underlying genetic and epigenetic contributions of neuronal function, behavior, and neurobiological diseases.
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Advances in CRISPR-Cas systems for epigenetics.
The CRISPR-Cas9 method has revolutionized the gene editing. Epigenetic changes, including DNA methylation, RNA modification, and changes in histone proteins, have been intensively studied and found to play a key role in the pathogenesis of human diseases. CRISPR-While the utility of DNA and chromatin modifications, known as epigenetics, is well understood, the functional significance of various alterations of RNA nucleotides has recently gained attention. Recent advancements in improving CRISPR-based epigenetic modifications has resulted in the availability of a powerful source that can selectively modify DNA, allowing for the maintenance of epigenetic memory over several cell divisions. Accurate identification of DNA methylation at specific locations is crucial for the prompt detection of cancer and other diseases, as DNA methylation is strongly correlated to the onset as well as the advancement of such conditions. Genetic or epigenetic perturbations can disrupt the regulation of imprinted genes, resulting in the development of diseases. When histone code editors and DNA de-/ methyltransferases are coupled with catalytically inactive Cas9 (dCas9), and CRISPRa and CRISPRi, they demonstrate excellent efficacy in editing the epigenome of eukaryotic cells. Advancing and optimizing the extracellular delivery platform can, hence, further facilitate the manipulation of CRISPR-Cas9 gene editing technique in upcoming clinical studies. The current chapter focuses on how the CRISP/ Cas9 system provides an avenue for the epigenetic modifications and its employability for human benefit.
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