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CRISPR-Cas Technology as a Revolutionary Genome Editing tool: Mechanisms and Biomedical Applications.
Programmable nucleases are powerful genomic tools for precise genome editing. These tools precisely recognize, remove, or change DNA at a defined site, thereby, stimulating cellular DNA repair pathways that can cause mutations or accurate replacement or deletion/insertion of a sequence. CRISPR-Cas9 system is the most potent and useful genome editing technique adapted from the defense immune system of certain bacteria and archaea against viruses and phages. In the past decade, this technology made notable progress, and at present, it has largely been used in genome manipulation to make precise gene editing in plants, animals, and human cells. In this review, we aim to explain the basic principle, mechanisms of action, and applications of this system in different areas of medicine, with emphasizing on the detection and treatment of parasitic diseases.
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Islamic Perspectives on CRISPR/Cas9-Mediated Human Germline Gene Editing: A Preliminary Discussion.
The recent development of CRISPR/Cas9 technology has rekindled the ethical debate concerning human germline modification that has begun decades ago. This inexpensive technology shows tremendous promise in disease prevention strategies, while raising complex ethical concerns about safety and efficacy of the technology, human dignity, tampering with God's creation, and human genetic enhancement. Germline gene editing may result in heritable changes in the human genome, therefore the question of whether it should be allowed requires deep and careful discussion from various perspectives. This paper explores Islamic perspectives on the concerns raised and highlights the ethical principles in Islam that should be taken into consideration when assessing the permissibility of CRISPR/ Cas9-mediated human germline gene editing. As argued in this paper, human germline gene editing would be considered lawful for medical purpose under certain conditions. It should not be applied on humans until the safety and efficacy issues are resolved. Robust ethical guidelines and strict regulations are necessary to preserve human dignity and to prevent premature and misuse of the technology. Maqasid al-shariah's principles of preservation of human life, lineage, and dignity and 'preventing harm takes precedence over securing benefit' are among the guiding principles in assessing the permissibility of CRISPR/Cas9-mediated human germline editing from an Islamic perspective. Further discussions are important to address the controversies as well as to explore the related ethical principles.
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Application of CRISPR in Filamentous Fungi and Macrofungi: From Component Function to Development Potentiality.
Fungi, particularly filamentous fungi and macrofungi, have a very powerful ability to produce secondary metabolites and can serve as excellent chassis cells for the production of enzymes or natural products of great value in synthetic biology. Thus, it is imperative to establish simple, reliable, and efficient techniques for their genetic modification. However, the heterokaryosis of some fungi and the dominance of nonhomologous end-joining (NHEJ) repair mechanisms in vivo have been greatly affecting the efficiency of fungal gene editing. In recent years, the CRISPR/Cas9 system has been applied as a widely used gene editing technology in life science research and has also played an important role in the genetic modification of filamentous and macrofungi. The various functional components (cas9, sgRNA, promoter, and screening marker) of the CRISPR/Cas9 system and its development, as well as the difficulties and potential of the CRISPR/Cas9 system in filamentous fungus and macrofungi, are the main topics of this paper.
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CRISPR-Cas9-mediated gene editing in human MPS I fibroblasts.
Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder (LSD). It is caused by mutations in the IDUA gene, which lead to the accumulation of the glycosaminoglycans dermatan and heparan sulfate. The CRISPR-Cas9 system is a new and powerful tool that allows gene editing at precise points of the genome, resulting in gene correction through the introduction and genomic integration of a wildtype sequence. In this study, we used the CRISPR-Cas9 genome editing technology to correct in vitro the most common mutation causing MPS I. Human fibroblasts homozygous for p.Trp402* (legacy name W402X) were transfected and analyzed for up to one month after treatment. IDUA activity was significantly increased, lysosomal mass was decreased, and next generation sequencing confirmed that a percentage of cells carried the wildtype sequence. As a proof of concept, this study demonstrates that CRISPR-Cas9 genome editing may be used to correct causative mutations in MPS I. LIST OF ABBREVIATIONS.
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The Ethics of Human Embryo Editing via CRISPR-Cas9 Technology: A Systematic Review of Ethical Arguments, Reasons, and Concerns.
The possibility of editing the genomes of human embryos has generated significant discussion and interest as a matter of science and ethics. While it holds significant promise to prevent or treat disease, research on and potential clinical applications of human embryo editing also raise ethical, regulatory, and safety concerns. This systematic review included 223 publications to identify the ethical arguments, reasons, and concerns that have been offered for and against the editing of human embryos using CRISPR-Cas9 technology. We identified six major themes: risk/harm; potential benefit; oversight; informed consent; justice, equity, and other social considerations; and eugenics. We explore these themes and provide an overview and analysis of the critical points in the current literature.
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Prospects and challenges of CRISPR/Cas9 gene-editing technology in cancer research.
Cancer, one of the leading causes of death, usually commences and progresses as a result of a series of gene mutations and dysregulation of expression. With the development of clustered regularly interspaced palindromic repeat (CRISPR)/Cas9 gene-editing technology, it is possible to edit and then decode the functions of cancer-related gene mutations, markedly advance the research of biological mechanisms and treatment of cancer. This review summarizes the mechanism and development of CRISPR/Cas9 gene-editing technology in recent years and describes its potential application in cancer-related research, such as the establishment of human tumor disease models, gene therapy and immunotherapy. The challenges and future development directions are highlighted to provide a reference for exploring pathological mechanisms and potential treatment protocols of cancer.
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Optically Controlled CRISPR-Cas9 and Cre Recombinase for Spatiotemporal Gene Editing: A Review.
CRISPR-Cas9 and Cre recombinase, two tools extensively used for genome interrogation, have catalyzed key breakthroughs in our understanding of complex biological processes and diseases. However, the immense complexity of biological systems and off-target effects hinder clinical applications, necessitating the development of platforms to control gene editing over spatial and temporal dimensions. Among the strategies developed for inducible control, light is particularly attractive as it is noninvasive and affords high spatiotemporal resolution. The principles for optical control of Cas9 and Cre recombinase are broadly similar and involve photocaged enzymes and small molecules, engineered split- and single-chain constructs, light-induced expression, and delivery by light-responsive nanocarriers. Few systems enable spatiotemporal control with a high dynamic range without loss of wild-type editing efficiencies. Such systems posit the promise of light-activatable systems in the clinic. While the prospect of clinical applications is palpably exciting, optimization and extensive preclinical validation are warranted. Judicious integration of optically activated CRISPR and Cre, tailored for the desired application, may help to bridge the "bench-to-bedside" gap in therapeutic gene editing.
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Gene Disruption Using CRISPR-Cas9 Technology.
The emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology provides tools for researchers to modify genomes in a specific and efficient manner. The Type II CRISPR-Cas9 system enables gene editing by directed DNA cleavage followed by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). Here, we described the use of the Type II CRISPR-Cas9 system in detail from designing the guides to analyzing the desired gene disruption events.
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Heritable Tissue-Culture-Free Gene Editing in Nicotiana benthamiana through Viral Delivery of SpCas9 and sgRNA.
Conventional plant gene editing requires laborious tissue-culture-mediated transformation, which restricts the range of applicable plant species. In this study, we developed a heritable and tissue-culture-free gene editing method in Nicotiana benthamiana using tobacco ringspot virus (TRSV) as a vector for in planta delivery of Cas9 and single-guide RNA (sgRNA) to shoot apical meristems. Agrobacterium-mediated inoculation of the TRSV vector induced systemic and heritable gene editing in Nicotiana benthamiana PHYTOENE DESATURASE. Transient downregulation of RNA silencing enhanced gene editing efficiency, resulting in an order of magnitude increase (0.8-13.2%) in the frequency of transgenerational gene editing. While the TRSV system had a preference for certain sgRNA sequences, co-inoculation of a TRSV vector carrying only Cas9 and a tobacco rattle virus vector carrying sgRNA successfully introduced systemic mutations with all five tested sgRNAs. Extensively gene-edited lateral shoots occasionally grew from plants inoculated with the virus vectors, the transgenerational gene editing frequency of which ranged up to 100%. This virus-mediated heritable gene editing method makes plant gene editing easy, requiring only the inoculation of non-transgenic plants with a virus vector(s) to obtain gene-edited individuals.
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Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9.
Single-stranded DNA (ssDNA) templates along with Cas9 have been used for knocking-in exogenous sequences in the genome but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 12-19% of internal bases, which we denote as enhanced ssDNA (esDNA), improve knock-in (KI) by 2-3-fold compared to end-modified ssDNA in airway basal stem cells (ABCs), CD34 + hematopoietic cells (CD34 + cells), T-cells and endothelial cells. Over 50% of alleles showed KI in three clinically relevant loci (CFTR, HBB and CCR5) in ABCs using esDNA and up to 70% of alleles showed KI in the HBB locus in CD34 + cells in the presence of a DNA-PKcs inhibitor. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. The esDNA templates did not improve KI in induced pluripotent stem cells (iPSCs). This may be due to the absence of the nuclease TREX1 in iPSCs. Indeed, knocking out TREX1 in other cells improved KI using unmodified ssDNA. esDNA can be used to modify 20-30 bp regions in primary cells for therapeutic applications and biological modeling. The use of this approach for gene length insertions will require new methods to produce long chemically modified ssDNA in scalable quantities.
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