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The Evolution of Nucleic Acid-Based Diagnosis Methods from the (pre-)CRISPR to CRISPR era and the Associated Machine/Deep Learning Approaches in Relevant RNA Design.
Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains.
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New Approaches for Targeting PCSK9: Small-Interfering Ribonucleic Acid and Genome Editing.
There is overwhelming clinical and genetic evidence supporting the concept that low-density-lipoprotein cholesterol should be as low as possible for as long as possible in patients at very high cardiovascular risk. Despite the wide availability of effective lipid-lowering therapies, the majority of patients still fail to reach guideline-based lipid goals. Advances in novel approaches targeting PCSK9 (proprotein convertase subtilisin/kexin type 9) through small-interfering RNA and genome editing hold the potential to bridge this gap, by offering long-acting alternatives, which may overcome adherence and other challenges in the current chronic care model. In this review, we discuss the history of targeting PCSK9 with the use of mRNA and small-interfering ribonucleic acid. We also shed light on targeting PCSK9 with genome editing, including discussion of the VERVE-101 clustered regularly interspaced short palindromic repeats-base editing medicine currently being evaluated in a clinical trial and others in development.
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Harnessing the native type I-B CRISPR-Cas for genome editing in a polyploid archaeon.
Research on CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated protein) systems has led to the revolutionary CRISPR/Cas9 genome editing technique. However, for most archaea and half of bacteria, exploitation of their native CRISPR-Cas machineries may be more straightforward and convenient. In this study, we harnessed the native type I-B CRISPR-Cas system for precise genome editing in the polyploid haloarchaeon Haloarcula hispanica. After testing different designs, the editing tool was optimized to be a single plasmid that carries both the self-targeting mini-CRISPR and a 600-800 bp donor. Significantly, chromosomal modifications, such as gene deletion, gene tagging or single nucleotide substitution, were precisely introduced into the vast majority of the transformants. Moreover, we showed that simultaneous editing of two genomic loci could also be readily achieved by one step. In summary, our data demonstrate that the haloarchaeal CRISPR-Cas system can be harnessed for genome editing in this polyploid archaeon, and highlight the convenience and efficiency of the native CRISPR-based genome editing strategy.
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In vivo CRISPR editing for cancer immunotherapy.
Cancer immunotherapy has shown significant promise in certain patient populations, but further advancements are needed to extend its benefits to a wider range of patients. Clustered regularly interspaced short palindromic repeats (CRISPR)-based editing has rapidly evolved in recent years, enabling its transition into direct therapeutic applications. This review summarizes recent progress in applying CRISPR systems in vivo for cancer immunotherapy, focusing on approaches that target cancer cells and the tumor microenvironment, as well as those that directly engineer immune cell populations themselves. Novel CRISPR editing platforms and strategies enabling multiplexed editing have also recently demonstrated promising impacts on driving antitumor immunity, however, the platforms investigated are still in the early stages and further investigation will be needed to robustly assess the potential for clinical translation. Future work can expand the array of therapeutic targets by incorporating data from functional genomics and must also carefully evaluate both editing modalities and delivery systems to optimize efficacy, safety, and scalability.
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CRISPR-Cas system to discover host-virus interactions in Flaviviridae.
The Flaviviridae virus family members cause severe human diseases and are responsible for considerable mortality and morbidity worldwide. Therefore, researchers have conducted genetic screens to enhance insight into viral dependency and develop potential anti-viral strategies to treat and prevent these infections. The host factors identified by the clustered regularly interspaced short palindromic repeats (CRISPR) system can be potential targets for drug development. Meanwhile, CRISPR technology can be efficiently used to treat viral diseases as it targets both DNA and RNA. This paper discusses the host factors related to the life cycle of viruses of this family that were recently discovered using the CRISPR system. It also explores the role of immune factors and recent advances in gene editing in treating flavivirus-related diseases. The ever-increasing advancements of this technology may promise new therapeutic approaches with unique capabilities, surpassing the traditional methods of drug production and treatment.
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CRISPR/Cas9 in plants: at play in the genome and at work for crop improvement.
The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system uses single-guide RNAs for genome editing, making it a simple, robust, powerful tool for targeted gene mutagenesis, knockout and knock-in/replacement, as well as transcriptional regulation. Here, we review the working principles, components and potential modifications of CRISPR/Cas9 for efficient single and multiplex gene editing in plants. We also describe recent work that has used CRISPR/Cas9 to improve economically important traits in crop plants. Although the apparent ease of CRISPR/Cas9-mediated editing may make it appear as though scientists are merely playing with plant genomes, the combined power of CRISPR/Cas9 has enabled vital research to be completed in the battle toward optimization and adaptation of crop species, permitting crucial advances to be achieved in crop improvement.
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Targeted Gene Editing of Glia Maturation Factor in Microglia: a Novel Alzheimer's Disease Therapeutic Target.
Alzheimer's disease (AD) is a devastating, progressive neurodegenerative disorder that leads to severe cognitive impairment in elderly patients. Chronic neuroinflammation plays an important role in the AD pathogenesis. Glia maturation factor (GMF), a proinflammatory molecule discovered in our laboratory, is significantly upregulated in various regions of AD brains. We have previously reported that GMF is predominantly expressed in the reactive glial cells surrounding the amyloid plaques (APs) in the mouse and human AD brain. Microglia are the major source of proinflammatory cytokines and chemokines including GMF. Recently clustered regularly interspaced short palindromic repeats (CRISPR) based genome editing has been recognized to study the functions of genes that are implicated in various diseases. Here, we investigated if CRISPR-Cas9-mediated GMF gene editing leads to inhibition of GMF expression and suppression of microglial activation. Confocal microscopy of murine BV2 microglial cell line transduced with an adeno-associated virus (AAV) coexpressing Staphylococcus aureus (Sa) Cas9 and a GMF-specific guide RNA (GMF-sgRNA) revealed few cells expressing SaCas9 while lacking GMF expression, thereby confirming successful GMF gene editing. To further improve GMF gene editing efficiency, we developed lentiviral vectors (LVs) expressing either Streptococcus pyogenes (Sp) Cas9 or GMF-sgRNAs. BV2 cells cotransduced with LVs expressing SpCas9 and GMF-sgRNAs revealed reduced GMF expression and the presence of indels in the exons 2 and 3 of the GMF coding sequence. Lipopolysaccharide (LPS) treatment of GMF-edited cells led to reduced microglial activation as shown by reduced p38 MAPK phosphorylation. We believe that targeted in vivo GMF gene editing has a significant potential for developing a unique and novel AD therapy.
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Cornerstones of CRISPR-Cas in drug discovery and therapy.
The recent development of CRISPR-Cas systems as easily accessible and programmable tools for genome editing and regulation is spurring a revolution in biology. Paired with the rapid expansion of reference and personalized genomic sequence information, technologies based on CRISPR-Cas are enabling nearly unlimited genetic manipulation, even in previously difficult contexts, including human cells. Although much attention has focused on the potential of CRISPR-Cas to cure Mendelian diseases, the technology also holds promise to transform the development of therapies to treat complex heritable and somatic disorders. In this Review, we discuss how CRISPR-Cas can affect the next generation of drugs by accelerating the identification and validation of high-value targets, uncovering high-confidence biomarkers and developing differentiated breakthrough therapies. We focus on the promises, pitfalls and hurdles of this revolutionary gene-editing technology, discuss key aspects of different CRISPR-Cas screening platforms and offer our perspectives on the best practices in genome engineering.
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CRISPR/Cas9 a simple, inexpensive and effective technique for gene editing.
In recent years, the number of tools and techniques that enable genetic material to be added, removed or altered at specific locations in the genome has increased significantly. The objective is to know the structure of genomes, the function of genes and improve gene therapy.In this work we intend to explain the functioning of the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) and the advantages that this technique may have compared to previously developed techniques, such as RNA interference (RNAi), Zinc Finger Nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) in gene and genome editing.We will start with the story of the discovery, then its biological function in the adaptive immune system of bacteria against bacteriophage attack, and ending with a description of the mechanism of action and its use in gene editing. We will also discuss other Cas enzymes with great potential for use in genome editing as an alternative to Cas9.CRISPR/Cas9 is a simple, inexpensive, and effective technique for gene editing with multiple applications from the development of functional genomics and epigenetics. This technique will, in the near future, have great applications in the development of cell models for use in medical and pharmaceutical processes, in targeted therapy, and improvement of agricultural and environmental species.
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Crystal structure of the anti-CRISPR, AcrIIC4.
Clustered regularly interspaced short palindromic repeats (CRISPRs)-CRISPR-associated protein systems are bacterial and archaeal defense mechanisms against invading elements such as phages and viruses. To overcome these defense systems, phages and viruses have developed inhibitors called anti-CRISPRs (Acrs) that are capable of inhibiting the host CRISPR-Cas system via different mechanisms. Although the inhibitory mechanisms of AcrIIC1, AcrIIC2, and AcrIIC3 have been revealed, the inhibitory mechanisms of AcrIIC4 and AcrIIC5 have not been fully understood and structural data are unavailable. In this study, we elucidated the crystal structure of Type IIC anti-CRISPR protein, AcrIIC4. Our structural analysis revealed that AcrIIC4 exhibited a helical bundle fold comprising four helixes. Further biochemical and biophysical analyses showed that AcrIIC4 formed a monomer in solution, and monomeric AcrIIC4 directly interacted with Cas9 and Cas9/sgRNA complex. Discovery of the structure of AcrIIC4 and their interaction mode on Cas9 will help us elucidate the diversity in the inhibitory mechanisms of the Acr protein family.
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