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Polyethylenimine based magnetic nanoparticles mediated non-viral CRISPR/Cas9 system for genome editing.
Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9) system has become a revolutionary tool for gene editing. Since viral delivery systems have significant side effects, and naked DNA delivery is not an option, the nontoxic, non-viral delivery of CRISPR/Cas9 components would significantly improve future therapeutic delivery. In this study, we aim at characterizing nanoparticles to deliver plasmid DNA encoding for the CRISPR-Cas system in eukaryotic cells in vitro. CRISPR/Cas9 complexed polyethylenimine (PEI) magnetic nanoparticles (MNPs) were generated. We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to evaluate efficient homology- directed repair (HDR) and non-homologous end joining (NHEJ) events following transfection with NPs. MNPs have been synthesized by co-precipitation with the average particle size around 20 nm in diameter. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. Genome editing events were as efficient as compared to standard lipofectamine transfection. Our approach tested non-viral delivery of CRISPR/Cas9 and DNA template to perform HDR and NHEJ in the same assay. We demonstrated that PEI-MNPs is a promising delivery system for plasmids encoding CRISPR/Cas9 and template DNA and thus can improve safety and utility of gene editing.
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CRISPR RNA-guided activation of endogenous human genes.
Short guide RNAs (gRNAs) can direct catalytically inactive CRISPR-associated 9 nuclease (dCas9) to repress endogenous genes in bacteria and human cells. Here we show that single or multiple gRNAs can direct dCas9 fused to a VP64 transcriptional activation domain to increase expression of endogenous human genes. This proof-of-principle work shows that clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems can target heterologous effector domains to endogenous sites in human cells.
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Current approaches in CRISPR-Cas9 mediated gene editing for biomedical and therapeutic applications.
A single gene mutation can cause a number of human diseases that affect the quality of life. Until the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) systems, it was challenging to correct a gene mutation to avoid a disease by reverting phenotypes. The advent of CRISPR technology has changed the field of gene editing, given its simplicity and intrinsic programmability, surpassing the limitations of both zinc-finger nuclease and transcription activator-like effector nuclease and becoming the method of choice for therapeutic gene editing by overcoming the bottlenecks of conventional gene-editing techniques. Currently, there is no commercially available medicinal cure to correct a gene mutation that corrects and reverses the abnormality of a gene's function. Devising reprogramming strategies for faithful recapitulation of normal phenotypes is a crucial aspect for directing the reprogrammed cells toward clinical trials. The CRISPR-Cas9 system has been promising as a tool for correcting gene mutations in maladies including blood disorders and muscular degeneration as well as neurological, cardiovascular, renal, genetic, stem cell, and optical diseases. In this review, we highlight recent developments and utilization of the CRISPR-Cas9 system in correcting or generating gene mutations to create model organisms to develop deeper insights into diseases, rescue normal gene functionality, and curb the progression of a disease. Delivery of CRISPR-components being a pivotal aspect in proving its effectiveness, various proven delivery systems have also been briefly discussed.
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CRISPR/Cas9-mediated knockout strategies for enhancing immunotherapy in breast cancer.
Breast cancer, a prevalent disease with significant mortality rates, often presents treatment challenges due to its complex genetic makeup. This review explores the potential of combining Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene knockout strategies with immunotherapeutic approaches to enhance breast cancer treatment. The CRISPR/Cas9 system, renowned for its precision in inducing genetic alterations, can target and eliminate specific cancer cells, thereby minimizing off-target effects. Concurrently, immunotherapy, which leverages the immune system's power to combat cancer, has shown promise in treating breast cancer. By integrating these two strategies, we can potentially augment the effectiveness of immunotherapies by knocking out genes that enable cancer cells to evade the immune system. However, safety considerations, such as off-target effects and immune responses, necessitate careful evaluation. Current research endeavors aim to optimize these strategies and ascertain the most effective methods to stimulate the immune response. This review provides novel insights into the integration of CRISPR/Cas9-mediated knockout strategies and immunotherapy, a promising avenue that could revolutionize breast cancer treatment as our understanding of the immune system's interplay with cancer deepens.
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A comprehensive review on the current status of CRISPR based clinical trials for rare diseases.
A considerable fraction of population in the world suffers from rare diseases. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its related Cas proteins offer a modern form of curative gene therapy for treating the rare diseases. Hereditary transthyretin amyloidosis, hereditary angioedema, duchenne muscular dystrophy and Rett syndrome are a few examples of such rare diseases. CRISPR/Cas9, for example, has been used in the treatment of β-thalassemia and sickle cell disease (Frangoul et al., 2021; Pavani et al., 2021) [1,2]. Neurological diseases such as Huntington's have also been focused in some studies involving CRISPR/Cas (Yang et al., 2017; Yan et al., 2023) [3,4]. Delivery of these biologicals via vector and non vector mediated methods depends on the type of target cells, characteristics of expression, time duration of expression, size of foreign genetic material etc. For instance, retroviruses find their applicability in case of ex vivo delivery in somatic cells due to their ability to integrate in the host genome. These have been successfully used in gene therapy involving X-SCID patients although, incidence of inappropriate activation has been reported. On the other hand, ex vivo gene therapy for β-thalassemia involved use of BB305 lentiviral vector for high level expression of CRISPR biological in HSCs. The efficacy and safety of these biologicals will decide their future application as efficient genome editing tools as they go forward in further stages of human clinical trials. This review focuses on CRISPR/Cas based therapies which are at various stages of clinical trials for treatment of rare diseases and the constraints and ethical issues associated with them.
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RNA-based therapies in liver metabolic diseases.
RNA-based therapeutics have rapidly emerged over the past decade, offering a new class of medicines that differ significantly from conventional drugs. These therapies can be programmed to target or restore defective genes, allowing for more personalised treatments and reducing side effects. Notably, RNA therapies have made significant progress in the treatment of genetic liver diseases, exemplified by small interfering RNA treatments for hereditary transthyretin amyloidosis, which use liver-targeting strategies such as GalNAc conjugation to improve efficacy and safety. RNA-based gene-editing technologies, such as base editor and prime editor clustered regularly interspaced short palindromic repeats systems, also show promise with their ability to minimise genomic rearrangements and cancer risk. While RNA therapies offer high precision, challenges remain in optimising delivery methods and ensuring long-term safety and efficacy. Lipid nanoparticle-mRNA therapeutics, particularly for protein replacement in rare diseases, have gained support from preclinical successes. Compared with viral gene therapies, mRNA therapies present a safer profile with reduced risks of genomic integration and oncogene activation. However, clinical trials, especially for rare diseases, face limitations such as small sample sizes and short observation periods. Further preclinical studies, including non-human primates, will be essential for refining trial designs. Despite their potential, the high costs of RNA therapies pose a challenge that will require cost-utility models to guide pricing and accessibility. Here, we discuss the fundamental aspects of RNA-based therapeutics and showcase the most relevant preclinical and clinical developments in genetic liver metabolic diseases.
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Optimal LentiCRISPR-Based System for Sequential CRISPR/Cas9 Screens.
The advent of genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening has advanced the understanding of molecular systems within cells. Here, we demonstrate the utility of sequentially performed CRISPR knockout screens that use an existing library to explore a biological question across the human genome, and then the remaining cells are used to examine each gene candidate against one common gene of interest. We call this approach "Many vs One" CRISPR screening, made possible by a modified 7SK promoter in place of the U6 promoter to drive expression of a single guide RNA. Inserting this novel 7SK promoter into the ubiquitously used lentiCRISPRv2 backbone is crucial, because it overcomes the need for a substantial increase in CRISPR library coverage during screening, sample processing, and next generation sequencing. This new 7SK vector equals the original lentiCRISPRv2 in lentiviral titer, knockout efficiency, and ease of use.
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Artificial intelligence and synthetic biology approaches for human gut microbiome.
The gut microbiome comprises a variety of microorganisms whose genes encode proteins to carry out crucial metabolic functions that are responsible for the majority of health-related issues in human beings. The advent of the technological revolution in artificial intelligence (AI) assisted synthetic biology (SB) approaches will play a vital role in the modulating the therapeutic and nutritive potential of probiotics. This can turn human gut as a reservoir of beneficial bacterial colonies having an immense role in immunity, digestion, brain function, and other health benefits. Hence, in the present review, we have discussed the role of several gene editing tools and approaches in synthetic biology that have equipped us with novel tools like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) systems to precisely engineer probiotics for diagnostic, therapeutic and nutritive value. A brief discussion over the AI techniques to understand the metagenomic data from the healthy and diseased gut microbiome is also presented. Further, the role of AI in potentially impacting the pace of developments in SB and its current challenges is also discussed. The review also describes the health benefits conferred by engineered microbes through the production of biochemicals, nutraceuticals, drugs or biotherapeutics molecules etc. Finally, the review concludes with the challenges and regulatory concerns in adopting synthetic biology engineered microbes for clinical applications. Thus, the review presents a synergistic approach of AI and SB toward human gut microbiome for better health which will provide interesting clues to researchers working in the area of rapidly evolving food and nutrition science.
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Application of Upstream Open Reading Frames (uORFs) Editing for the Development of Stress-Tolerant Crops.
Global population growth and climate change are posing increasing challenges to the production of a stable crop supply using current agricultural practices. The generation of genetically modified (GM) crops has contributed to improving crop stress tolerance and productivity; however, many regulations are still in place that limit their commercialization. Recently, alternative biotechnology-based strategies, such as gene-edited (GE) crops, have been in the spotlight. Gene-editing technology, based on the clustered regularly interspaced short palindromic repeats (CRISPR) platform, has emerged as a revolutionary tool for targeted gene mutation, and has received attention as a game changer in the global biotechnology market. Here, we briefly introduce the concept of upstream open reading frames (uORFs) editing, which allows for control of the translation of downstream ORFs, and outline the potential for enhancing target gene expression by mutating uORFs. We discuss the current status of developing stress-tolerant crops, and discuss uORF targets associated with salt stress-responsive genes in rice that have already been verified by transgenic research. Finally, we overview the strategy for developing GE crops using uORF editing via the CRISPR-Cas9 system. A case is therefore made that the mutation of uORFs represents an efficient method for developing GE crops and an expansion of the scope of application of genome editing technology.
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CRISPR/Cas as a Genome-Editing Technique in Fruit Tree Breeding.
CRISPR (short for "Clustered Regularly Interspaced Short Palindromic Repeats") is a technology that research scientists use to selectively modify the DNA of living organisms. CRISPR was adapted for use in the laboratory from the naturally occurring genome-editing systems found in bacteria. In this work, we reviewed the methods used to introduce CRISPR/Cas-mediated genome editing into fruit species, as well as the impacts of the application of this technology to activate and knock out target genes in different fruit tree species, including on tree development, yield, fruit quality, and tolerance to biotic and abiotic stresses. The application of this gene-editing technology could allow the development of new generations of fruit crops with improved traits by targeting different genetic segments or even could facilitate the introduction of traits into elite cultivars without changing other traits. However, currently, the scarcity of efficient regeneration and transformation protocols in some species, the fact that many of those procedures are genotype-dependent, and the convenience of segregating the transgenic parts of the CRISPR system represent the main handicaps limiting the potential of genetic editing techniques for fruit trees. Finally, the latest news on the legislation and regulations about the use of plants modified using CRISPR/Cas systems has been also discussed.
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