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A comprehensive overview of computational resources to aid in precision genome editing with engineered nucleases.
Genome editing with engineered nucleases (zinc finger nucleases, TAL effector nucleases s and Clustered regularly inter-spaced short palindromic repeats/CRISPR-associated) has recently been shown to have great promise in a variety of therapeutic and biotechnological applications. However, their exploitation in genetic analysis and clinical settings largely depends on their specificity for the intended genomic target. Large and complex genomes often contain highly homologous/repetitive sequences, which limits the specificity of genome editing tools and could result in off-target activity. Over the past few years, various computational approaches have been developed to assist the design process and predict/reduce the off-target activity of these nucleases. These tools could be efficiently used to guide the design of constructs for engineered nucleases and evaluate results after genome editing. This review provides a comprehensive overview of various databases, tools, web servers and resources for genome editing and compares their features and functionalities. Additionally, it also describes tools that have been developed to analyse post-genome editing results. The article also discusses important design parameters that could be considered while designing these nucleases. This review is intended to be a quick reference guide for experimentalists as well as computational biologists working in the field of genome editing with engineered nucleases.
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Nature Inspired Delivery Vehicles for CRISPR-Based Genome Editing.
The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genome editing technologies has opened up groundbreaking possibilities for treating a wide spectrum of genetic disorders and diseases. However, the success of these technologies relies heavily on the development of efficient and safe delivery systems. Among the most promising approaches are natural and synthetic nanocarrier-mediated delivery systems, including viral vectors, extracellular vesicles (EVs), engineered cellular membrane particles, liposomes, and various nanoparticles. These carriers enhance the efficacy of the CRISPR system by providing a unique combination of efficiency, specificity, and reduced immunogenicity. Synthetic carriers such as liposomes and nanoparticles facilitate CRISPR delivery with high reproducibility and customizable functions. Viral vectors, renowned for their high transduction efficiency and broad tropism, serve as powerful vehicles for delivering CRISPR components to various cell types. EVs, as natural carriers of RNA and proteins, offer a stealth mechanism to evade immune detection, allowing for the targeted delivery of genome editors with minimal off-target effects. Engineered cellular membrane particles further improve delivery by simulating the cellular environment, enhancing uptake, and minimizing immune response. This review explores the innovative integration of CRISPR genome editors with various nanocarrier systems, focusing on recent advancements, applications, and future directions in therapeutic genome editing.
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Vectors in CRISPR Gene Editing for Neurological Disorders: Challenges and Opportunities.
Diseases of the nervous system are recognized as the second leading cause of death worldwide. The global prevalence of neurological diseases, such as Huntington's disease, Alzheimer's disease, and Parkinson's disease has seen a significant rise due to the increasing proportion of the aging population. The discovery of the clustered regularly interspaced short palindromic repeats (CRISPR) genome editing technique has paved way for universal neurological diseases treatment. However, finding a safe and effective method to deliver CRISPR gene-editing tools remains a main challenge for genome editing therapies in vivo. Adeno-associated virus (AAV) is currently one of the most commonly used vector systems, but some issues remain unresolved, including capsid immunogenicity, off-target mutations, and potential genotoxicity. To address these concerns, researchers are actively encouraging the development of new delivery systems, like virus-like particles and nanoparticles. These novel systems have the potential to enhance targeting efficiency, thereby offering possible solutions to the current challenges. This article reviews CRISPR delivery vectors for neurological disorders treatment and explores potential solutions to overcome limitations in vector systems. Additionally, the delivery strategies of CRISPR systems are highlighted as valuable tools for studying neurological diseases, and the challenges and opportunities that these vectors present.
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CRISPR-Based genome editing in pulses: current approaches, challenges, and future prospects.
Legumes are the second most important food crop after cereals for the world population. It is a significant protein source for developing countries and integral to global food security. However, various agroecological constraints and biotic and abiotic factors often compromise the production of pulses. Legumes are long-term neglected crops worldwide and follow traditional breeding, leading to a time-consuming, labor-intensive, less economically feasible program associated with linkage drag. Recent sequencing attempts in the twenty-first century, with the development of an enormous repertoire of genetic and genomic resources, allowed scientists to accelerate the improvement of legumes with modern genome editing tools. One such promising tool is CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which has revolutionized and transformed the landscape of genetic engineering. The emergence of CRISPR/Cas systems has redefined precision breeding, offering unprecedented control over genome manipulation in legume crops. It has tremendous potential for crop improvement and can precisely make changes at genomic locations with incredible accuracy. Therefore, identifying the desired genes and their precise manipulation has enormous implications for legume crop improvement. This review will give an overview of the genome editing tools available for crop improvement and the efficiency of different transformation methods in legume crops. It will also discuss the current status of genome editing in legume crops, including challenges and future perspectives.
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Yeast genome, by design.
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Application of CRISPR/Cas9-Based Gene Editing in HIV-1/AIDS Therapy.
Despite the fact that great efforts have been made in the prevention and therapy of HIV-1 infection, HIV-1/AIDS remains a major threat to global human health. Highly active antiretroviral therapy (HAART) can suppress virus replication, but it cannot eradicate latent viral reservoirs in HIV-1/AIDS patients. Recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has been engineered as an effective gene-editing technology with the potential to treat HIV-1/AIDS. It can be used to target cellular co-factors or HIV-1 genome to reduce HIV-1 infection and clear the provirus, as well as to induce transcriptional activation of latent virus in latent viral reservoirs for elimination. This versatile gene editing technology has been successfully applied to HIV-1/AIDS prevention and reduction in human cells and animal models. Here, we update the rapid progress of CRISPR/Cas9-based HIV-1/AIDS therapy research in recent years and discuss the limitations and future perspectives of its application.
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Tighten up Mexico's regulations on human gene editing.
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Delivery of CRISPR/Cas systems for cancer gene therapy and immunotherapy.
The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems are efficient and versatile gene editing tools, which offer enormous potential to treat cancer by editing genome, transcriptome or epigenome of tumor cells and/or immune cells. A large body of works have been done with CRISPR/Cas systems for genetic modification, and 16 clinical trials were conducted to treat cancer by ex vivo or in vivo gene editing approaches. Now, promising preclinical works have begun using CRISPR/Cas systems in vivo. However, efficient and safe delivery of CRISPR/Cas systems in vivo is still a critical challenge for their clinical applications. This article summarizes delivery of CRISPR/Cas systems by physical methods, viral vectors and non-viral vectors for cancer gene therapy and immunotherapy. The prospects for the development of physical methods, viral vectors and non-viral vectors for delivery of CRISPR/Cas systems are reviewed, and promising advances in cancer treatment using CRISPR/Cas systems are discussed.
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CRISPR tools for T cells: targeting the genome, epigenome, and transcriptome.
T cell therapy has curative potential for many cancers. Despite impressive clinical efficacy in hematological malignancies, current T cell therapy still faces challenges related to sustaining responses, antigen escape, cytotoxicity, limited accessibility, and difficulties in treating solid tumors. The advent of CRISPR (clustered regularly interspaced short palindromic repeats) technologies provides a promising solution to these challenges. CRISPR technologies have grown from merely tools for gene knockout to sophisticated tools that can engineer cells at various levels of the genome, epigenome, and transcriptome. In this review we discuss recent technological advancements and how their application to T cells has the potential to steer the next generation of cellular therapy. We highlight emerging applications and current technological limitations that future tool development aims to overcome.
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Genetically modified crop regulations: scope and opportunity using the CRISPR-Cas9 genome editing approach.
Global demand for food is increasing day by day due to an increase in population and shrinkage of the arable land area. To meet this increasing demand, there is a need to develop high-yielding varieties that are nutritionally enriched and tolerant against environmental stresses. Various techniques are developed for improving crop quality such as mutagenesis, intergeneric crosses, and translocation breeding. Later, with the development of genetic engineering, genetically modified crops came up with the transgene insertion approach which helps to withstand adverse conditions. The process or product-focused approaches are used for regulating genetically modified crops with their risk analysis on the environment and public health. However, recent advances in gene-editing technologies have led to a new era of plant breeding by developing techniques including site-directed nucleases, zinc finger nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) that involve precise gene editing without the transfer of foreign genes. But these techniques always remain in debate for their regulation status and public acceptance. The European countries and New Zealand, consider the gene-edited plants under the category of genetically modified organism (GMO) regulation while the USA frees the gene-edited plants from such type of regulations. Considering them under the category of GMO makes a long and complicated approval process to use them, which would decrease their immediate commercial value. There is a need to develop strong regulatory approaches for emerging technologies that expedite crop research and attract people to adopt these new varieties without hesitation.
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