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Gene Editing and Human Pluripotent Stem Cells: Tools for Advancing Diabetes Disease Modeling and Beta-Cell Development.
PURPOSE OF REVIEW: This review will focus on the multiple approaches to gene editing and address the potential use of genetically modified human pluripotent stem cell-derived beta cells (SC-β) as a tool to study human beta-cell development and model their function in diabetes. We will explore how new variations of CRISPR/Cas9 gene editing may accelerate our understanding of beta-cell developmental biology, elucidate novel mechanisms that establish and regulate beta-cell function, and assist in pioneering new therapeutic modalities for treating diabetes. RECENT FINDINGS: Improvements in CRISPR/Cas9 target specificity and homology-directed recombination continue to advance its use in engineering stem cells to model and potentially treat disease. We will review how CRISPR/Cas9 gene editing is informing our understanding of beta-cell development and expanding the therapeutic possibilities for treating diabetes and other diseases. Here we focus on the emerging use of gene editing technology, specifically CRISPR/Cas9, as a means of manipulating human gene expression to gain novel insights into the roles of key factors in beta-cell development and function. Taken together, the combined use of SC-β cells and CRISPR/Cas9 gene editing will shed new light on human beta-cell development and function and accelerate our progress towards developing new therapies for patients with diabetes.
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CRISPR-Cas9-Guided Genetic Manipulation of Patient-Derived Brain Tumor Cells.
CRISPR-Cas9 technology has revolutionized scientific research and has provided scientists with the ability to change DNA bases specifically and precisely at predetermined sites. The CRISPR-Cas9 knockout (KO) and activation (a) platforms developed by Hart et al. and Sanson et al. allow for RNA-directed genome editing to both decrease or increase gene expression, respectively [1, 2]. In this chapter the two techniques, CRISPR KO and CRISPRa, will be discussed and explained in further detail for optimized application against patient derived brain tumor cells.
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Biomaterial-assisted targeted and controlled delivery of CRISPR/Cas9 for precise gene editing.
RISPR-Cas9 has exhibited enormous potential in gene therapy. It can perform genome editing with single-nucleotide precision in various types of cell and tissue, providing a powerful breakthrough technology for genome editing in therapeutic development. But the limited delivery methods pose substantial challenges pertinent to safe and effective CRISPR/Cas9 delivery, thus hindering its application. These challenges should be tackled to develop next-generation genetic therapies. Biomaterial-based drug delivery systems can overcome these issues, for example using biomaterials as carriers for CRISPR/Cas9 targeted delivery, and conditional control of its function can improve precision, furnish on-demand and transient gene editing and reduce adverse consequences such as off-target events and immunogenicity, representing a promising direction for modern precision medicine. This review describes the application status and research progress of current CRISPR/Cas9 delivery approaches, including polymeric nanoparticles, liposomes, extracellular vesicles, inorganic nanoparticles and hydrogels. The unique properties of light-controlled and small-molecule drugs for spatially and temporally controlled genome editing are also illustrated. In addition, targetable delivery vehicles for the active delivery of CRISPR systems are also discussed. The perspectives to overcome the current limitations in the CRISPR/Cas9 delivery and their bench-to-bedside translation are also highlighted.
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In Silico Design of gRNA for CRISPR/Cas9-Mediated Gene Knockout.
CRISPR/Cas9 stands as a revolutionary and versatile gene editing technology. At its core, the Cas9 DNA endonuclease is guided with precision by a specifically designed single-guide RNA (gRNA). This guidance system facilitates the introduction of double-stranded breaks (DSBs) within the DNA. Subsequent imprecise repairs, mainly through the non-homologous end-joining (NHEJ) pathway, yield insertions or deletions, resulting in frameshift mutations. These mutations are instrumental in achieving the successful knockout of the target gene. In this chapter, we describe all necessary steps to create and design a gRNA for a gene knockout to a target gene before to transfer it to a target plant.
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Production of single- and multiple-gene-modified mice via maternal SpCas9-based gene editing.
Maternally and transiently accumulated SpCas9 (maternal SpCas9) in a zygote derived from a systemically SpCas9-expressing transgenic mouse strain was used to generate single- and multiple-gene-modified mice. Maternal SpCas9-based gene editing allows for high indel and knockin mutation efficiency, low mosaicism, increased pup delivery rate, and simultaneous induction of mutations at multiple loci in contrast to conventional CRISPR/SpCas9-based gene editing. For complete details on the use and execution of this protocol, please refer to Sakurai et al. (2020).
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CRISPR-Cas-based detection for food safety problems: Current status, challenges, and opportunities.
Food safety is one of the biggest public issues occurring around the world. Microbiological, chemical, and physical hazards can lead to food safety issues, which may occur at all stages of the supply chain. In order to tackle food safety issues and safeguard consumer health, rapid, accurate, specific, and field-deployable detection methods meeting diverse requirements are one of the imperative measures for food safety assurance. CRISPR-Cas system, a newly emerging technology, has been successfully repurposed in biosensing and has demonstrated huge potential to establish conceptually novel detection methods with high sensitivity and specificity. This review focuses on CRISPR-Cas-based detection and its current status and huge potential specifically for food safety inspection. We firstly illustrate the pending problems in food safety and summarize the popular detection methods. We then describe the potential applications of CRISPR-Cas-based detection in food safety inspection. Finally, the challenges and futuristic opportunities are proposed and discussed. Generally speaking, the current food safety detection methods are still unsatisfactory in some ways such as being time-consuming, displaying unmet sensitivity and specificity standards, and there is a comparative paucity of multiplexed testing and POCT. Recent studies have shown that CRISPR-Cas-based biosensing is an innovative and fast-expanding technology, which could make up for the shortcomings of the existing methods or even replace them. To sum up, the implementation of CRISPR-Cas and the integration of CRISPR-Cas with other techniques is promising and desirable, which is expected to provide "customized" and "smart" detection methods for food safety inspection in the coming future.
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Efficient tobacco rattle virus-induced gene editing in tomato mediated by the CRISPR/Cas9 system.
Plant virus-based sgRNA delivery strategy has been widely applied for efficient genome editing across various plant species, leveraging its significant advantages in the rapid expression and expansion of sgRNA through virus replication and movement. However, the efficacy of the virus-induced gene editing (VIGE) tool in tomato has yet to be explored. In this paper, we established a TRV-mediated CRISPR/Cas9 genome editing system in the somatic cells of tomato, reporting the validation of VIGE and evaluating the mutagenesis efficiency in both tomato leaves and fruits using high-throughput sequencing. The results demonstrated an approximate 65% efficiency of VIGE in tomato leaves for the selected target genes, with VIGE efficiency reaching up to 50% in tomato fruits. This research not only introduces an efficient tool for reverse genetics but also reveals substantial potential of VIGE in surpassing traditional tissue culture techniques for creating heritable mutations in tomato.
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Generating Mutant Renal Cell Lines Using CRISPR Technologies.
Gene editing using the CRISPR/Cas9 system is an extremely efficient approach for generating mutations within the genomic DNA of immortalized cell lines. This procedure begins with a straightforward cloning step to generate a single plasmid encoding the Cas9 enzyme as well as a synthetic guide RNA (sgRNA) which is selected to target specific sites within the genome. This plasmid is transfected into cells either alone, in order to generate random insertion-deletion alleles ("indels") at the desired locus via the nonhomologous end-joining pathway, or in conjunction with a homology-directed repair template oligonucleotide to generate a specific point mutation. Here we describe a procedure to perform gene editing in IMCD3 and HEK293 cells and to subsequently isolate clonal cell lines carrying mutations of interest.
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Virus-Like Particles as a Nonviral CRISPR/Cas Editing Delivery Tool.
Virus-like particles (VLPs) are self-assembling viral protein complexes that mimic native virions while lacking replicative and infectious capacity. By retaining efficient cellular entry and cargo protection, they overcome key limitations of viral vectors, including insertional mutagenesis and strong immunogenicity. Their ease of production, flexible cargo capacity, and tunable tropism make them highly versatile tools. Here, we describe a protocol for generating VSV-G-pseudotyped VLPs that deliver CRISPR/Cas9 RNPs and their functional validation in surrogate cell lines.
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Directed Evolution of CRISPR/Cas Systems for Precise Gene Editing.
CRISPR technology is a universal tool for genome engineering that has revolutionized biotechnology. Recently identified unique CRISPR/Cas systems, as well as re-engineered Cas proteins, have rapidly expanded the functions and applications of CRISPR/Cas systems. The structures of Cas proteins are complex, containing multiple functional domains. These protein domains are evolutionarily conserved polypeptide units that generally show independent structural or functional properties. In this review, we propose using protein domains as a new way to classify protein engineering strategies for these proteins and discuss common ways to engineer key domains to modify the functions of CRISPR/Cas systems.
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