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[Current advances and future prospects of genome editing technology in the field of biomedicine.].
Genome editing technology can alter the genomic sequence at will, contributing the creation of cellular and animal models of human diseases including hereditary disorders and cancers, and the generation of the mutation-corrected human induced pluripotent stem cells for ex vivo regenerative medicine. In addition, novel approaches such as drug development using genome-wide CRISPR screening and cancer suppression using epigenome editing technology, which can change the epigenetic modifications in a site-specific manner, have also been conducted. In this article, I summarize the current advances and future prospects of genome editing technology in the field of biomedicine.
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Two Distinct Approaches for CRISPR-Cas9-Mediated Gene Editing in Cryptococcus neoformans and Related Species.
Cryptococcus neoformans and related species are encapsulated basidiomycetous fungi that cause meningoencephalitis in individuals with immune deficiency. This pathogen has a tractable genetic system; however, gene disruption via electroporation remains difficult, while biolistic transformation is often limited by lack of multiple genetic markers and the high initial cost of equipment. The approach using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) has become the technology of choice for gene editing in many organisms due to its simplicity, efficiency, and versatility. The technique has been successfully demonstrated in C. neoformans and Cryptococcus deneoformans in which two DNA plasmids expressing either the Streptococcus pyogenesCAS9 gene or the guide RNA (gRNA) were employed. However, potential adverse effects due to constitutive expression and the time-consuming process of constructing vectors to express each gRNA remain as a primary barrier for wide adaptation. This report describes the delivery of preassembled CRISPR-Cas9-gRNA ribonucleoproteins (RNPs) via electroporation that is able to generate edited mutant alleles. RNP-mediated CRISPR-Cas9 was used to replace the wild-type GIB2 gene encoding a Gβ-like/RACK1 Gib2 protein with a gib2::NAT allele via homologous recombination in both C. neoformans and C. deneoformans In addition, a DNA plasmid (pCnCas9:U6-gRNA) that expresses both Cas9 and gRNA, allowing for convenient yet low-cost DNA-mediated gene editing, is described. pCnCas9:U6-gRNA contains an endogenous U6 promoter for gRNA expression and restriction sites for one-step insertion of a gRNA. These approaches and resources provide new opportunities to accelerate genetic studies of Cryptococcus species.IMPORTANCE For genetic studies of the Cryptococcus genus, generation of mutant strains is often hampered by a limited number of selectable genetic markers, the tedious process of vector construction, side effects, and other limitations, such as the high cost of acquiring a particle delivery system. CRISPR-Cas9 technology has been demonstrated in Cryptococcus for genome editing. However, it remains labor-intensive and time-consuming since it requires the identification of a suitable type III RNA polymerase promoter for gRNA expression. In addition, there may be potential adverse effects caused by constitutive expressions of Cas9 and gRNA. Here, I report the use of a ribonucleoprotein-mediated CRISPR-Cas9 technique for genome editing of C. neoformans and related species. Together with the custom-constructed pCnCas9:U6-gRNA vector that allows low-cost and time-saving DNA-based CRISPR-Cas9, my approach adds to the molecular toolbox for dissecting the molecular mechanism of pathogenesis in this important group of fungal pathogens.
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CRISPR immunity: a case study for justified somatic genetic modification?
The current SARS-CoV-2 pandemic has killed thousands across the world. SARS-CoV-2 is the latest but surely not the last such global pandemic we will face. The biomedical response to such pandemics includes treatment, vaccination, and so on. In this paper, though, we argue that it is time to consider an additional strategy: the somatic (non-heritable) enhancement of human immunity. We argue for this approach and consider bioethics objections we believe can be overcome.
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Delivery methods for site-specific nucleases: Achieving the full potential of therapeutic gene editing.
The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications. However, safe and efficient delivery methods remain the major obstacle for therapeutic gene editing. In this review, we summarize the recent progress on nuclease delivery methods, highlight their impact on the outcomes of gene editing and discuss the potential of different delivery approaches for therapeutic gene editing.
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Combining Induced Pluripotent Stem Cells and Genome Editing Technologies for Clinical Applications.
In this review, we introduce current developments in induced pluripotent stem cells (iPSCs), site-specific nuclease (SSN)-mediated genome editing tools, and the combined application of these two novel technologies in biomedical research and therapeutic trials. The sustainable pluripotent property of iPSCs in vitro not only provides unlimited cell sources for basic research but also benefits precision medicines for human diseases. In addition, rapidly evolving SSN tools efficiently tailor genetic manipulations for exploring gene functions and can be utilized to correct genetic defects of congenital diseases in the near future. Combining iPSC and SSN technologies will create new reliable human disease models with isogenic backgrounds in vitro and provide new solutions for cell replacement and precise therapies.
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What Do We (Not) Know About Global Views of Human Gene Editing? Insights and Blind Spots in the CRISPR Era.
As research on human applications of CRISPR advances, researchers, advisory bodies, and other stakeholder organizations continue calling for global public discourses and engagement to shape the development of human gene editing (HGE). Research that captures public views and tests ways for engaging across viewpoints is vital for facilitating these discourses. Unfortunately, such research lags behind advances in HGE research and applications. Here, we provide the first review of nationally representative public-opinion surveys focused on HGE to discuss limitations and remaining gaps, illustrating how these gaps hinder interpretation of existing studies. Rigorous research with proper methods for capturing representative public opinion of HGE is limited, especially in countries outside of the United States and on a global scale. The result is severely restricted understanding of even the surface level of public views concerning HGE. We identify broad areas where we need more and better research capturing public views, and describe how future surveys can help collect insights necessary for discourse and decision making on HGE.
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Comparison of efficiency and specificity of CRISPR-associated (Cas) nucleases in plants: An expanded toolkit for precision genome engineering.
Molecular tools adapted from bacterial CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems for adaptive immunity have become widely used for plant genome engineering, both to investigate gene functions and to engineer desirable traits. A number of different Cas (CRISPR-associated) nucleases are now used but, as most studies performed to date have engineered different targets using a variety of plant species and molecular tools, it has been difficult to draw conclusions about the comparative performance of different nucleases. Due to the time and effort required to regenerate engineered plants, efficiency is critical. In addition, there have been several reports of mutations at sequences with less than perfect identity to the target. While in some plant species it is possible to remove these so-called 'off-targets' by backcrossing to a parental line, the specificity of genome engineering tools is important when targeting specific members of closely-related gene families, especially when recent paralogues are co-located in the genome and unlikely to segregate. Specificity is also important for species that take years to reach sexual maturity or that are clonally propagated. Here, we directly compare the efficiency and specificity of Cas nucleases from different bacterial species together with engineered variants of Cas9. We find that the nucleotide content of the target correlates with efficiency and that Cas9 from Staphylococcus aureus (SaCas9) is comparatively most efficient at inducing mutations. We also demonstrate that 'high-fidelity' variants of Cas9 can reduce off-target mutations in plants. We present these molecular tools as standardised DNA parts to facilitate their re-use.
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Energy biotechnology in the CRISPR-Cas9 era.
The production of bioenergy from plant biomass previously relied on using microorganisms that rapidly and efficiently convert simple sugars into fuels and chemicals. However, to exploit the far more abundant carbon fixed in plant cell walls, future industrial production hosts will need to be engineered to leverage the most efficient biochemical pathways and most robust traits that can be found in nature. The CRISPR-Cas9 genome editing technology now enables writing the genome at will, which will allow biotechnology to become an 'information science.' This review covers recent advances in using CRISPR-Cas9 to engineer the genomes of a wide variety of organisms that could be use in the industrial production of biofuels and renewable chemicals.
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Safety of Germline Genome Editing for Genetically Related "Future" Children as Perceived by Parents.
The social acceptability of germline genome editing (GGE) depends on its perceived safety, as well as respect for reproductive autonomy. However, it is doubtful that prospective parents sufficiently understand the risks of GGE. In the future, the use of GGE in specific situations seems plausible, as it offers couples potential means to safeguard genetically related future children from a serious disease and overcome infertility due to a gene mutation. Should GGE fail, however, some couples may be obliged to abort affected fetuses, or give birth to adversely affected children, which would be a tragedy. Some children might develop diseases later in life due to overlooked off-target mutations. Compounding this, some parents are unlikely to inform their offspring about the details of conception, hampering necessary follow-up. Prospective parents, scientists and policy makers should carefully discuss the safety implications of GGE for genetically related future children.
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CRISPR Crops: Plant Genome Editing Toward Disease Resistance.
Genome editing by sequence-specific nucleases (SSNs) has revolutionized biology by enabling targeted modifications of genomes. Although routine plant genome editing emerged only a few years ago, we are already witnessing the first applications to improve disease resistance. In particular, CRISPR-Cas9 has democratized the use of genome editing in plants thanks to the ease and robustness of this method. Here, we review the recent developments in plant genome editing and its application to enhancing disease resistance against plant pathogens. In the future, bioedited disease resistant crops will become a standard tool in plant breeding.
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