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Workflow for Performing Genetic Manipulation in Human Trophoblast Stem Cells Using CRISPR/Cas9 Technology.
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based tool kits greatly facilitate the investigation of gene function, especially gene editing and transcriptional regulation technology. Human trophoblast stem cell (hTSC) is a promising cell model for simulating the development and function of human placental trophoblast cells. Here, we provide an optimized workflow for efficient genome editing and transcriptional activation in hTSCs using a lentivirus-dependent method. We also present methods to evaluate the gene targeted mutations and transcriptional activation.
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Reducing CRISPR-Cas9 off-target effects by optically controlled chemical modifications of guide RNA.
A photocatalytic click chemistry approach, offering a significant advancement over conventional methods in RNA function modulation is described. This innovative method, utilizing light-activated small molecules, provides a high level of precision and control in RNA regulation, particularly effective in intricate cellular processes. By applying this strategy to CRISPR-Cas9 gene editing, we demonstrate its effectiveness in enhancing gene editing specificity and markedly reducing off-target effects. Our approach employs a vinyl ether modification in RNA, which activated under visible light with a phenanthrenequinone derivative, creating a CRISPR-OFF switch that precisely regulates CRISPR system activity. This method not only represents an advancement in genomic interventions but also offers broad applications in gene regulation, paving the way for safer and more reliable gene editing in therapeutic genomics.
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CRISPR-Cas9‑based lipid nanocarriers for advanced therapy of urinary bladder cancer.
Bladder cancer (BCa) exhibits significant genetic and phenotypic variability. This variability suggests that various tumor subtypes could be influenced by several biomarkers and signaling pathways, which presents a problem for monotherapy strategies. Despite the initial effectiveness of traditional therapies, BCa's high rates of progression and recurrence, and the eventual development of drug resistance in many patients, continue to be major concerns. Because of the potential to transform the genetic modifications linked to the disease, genome editing using CRISPR/Cas9 has become a transformative tool in medicine with noteworthy potential for BCa therapy. Although the CRISPR/Cas9 technology is incredibly effective at transforming the field of genome editing, its instability and cellular impermeability pose significant challenges to its delivery. To increase efficient delivery of CRISPR/Cas9, nanovectors may be investigated. Significant promise exists for improving the therapeutic potential of CRISPR-Cas9 technology and addressing complex cancer therapy difficulties because of the rapid development of nanotechnology-based delivery systems. Relevant articles were searched in Google Scholar, Scopus, and Web of Science covering studies published between 2007 and 2026. Along with the impact of lipid-based nanoparticles on their safe transport to cancer cells, this review emphasizes the significance of the CRISPR/Cas9 genome editing system in the treatment of BCa.
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Leveraging CRISPR/Cas9 for optimized adoptive T cell therapies: From molecular engineering to clinical manufacturing.
Cancer immunotherapy is rapidly evolving from pharmacologic immune modulation to adoptive cell therapy (ACT). In ACT, T cells are expanded and genetically engineered ex vivo to achieve long-lasting antitumor activity. The primary ACT platforms-tumor-infiltrating lymphocytes (TIL), chimeric antigen receptor (CAR) T cells, and T-cell receptor (TCR) T cells-rely on T-cell effector function but differ in their mechanisms of antigen recognition, HLA dependence, and engineering requirements, leading to unique clinical strengths and limitations. CRISPR/Cas9 genome editing provides precise knock-out (KO) and knock-in (KI) strategies, allowing for multiplex editing and functional modulation across the genome. In the context of ACT manufacturing, CRISPR/Cas9 addresses critical challenges such as T-cell exhaustion, graft-versus-host disease (GvHD), and ensuring product consistency and quality. This article explores how CRISPR/Cas9 can be utilized to overcome the limitations of ACT and summarizes the current clinical landscape of CRISPR-engineered ACT products. Finally, we discuss the ongoing challenges associated with CRISPR-based genome editing and propose potential solutions.
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Multiplex CRISPR/Cas9-mediated genome editing to address drought tolerance in wheat.
Genome editing tools have rapidly been adopted by plant scientists for crop improvement. Genome editing using a multiplex sgRNA-CRISPR/Cas9 genome editing system is a useful technique for crop improvement in monocot species. In this study, we utilized precise gene editing techniques to generate wheat 3'(2'), 5'-bisphosphate nucleotidase (TaSal1) mutants using a multiplex sgRNA-CRISPR/Cas9 genome editing system. Five active TaSal1 homologous genes were found in the genome of Giza168 in addition to another apparently inactive gene on chromosome 4A. Three gRNAs were designed and used to target exons 4, 5 and 7 of the five wheat TaSal1 genes. Among the 120 Giza168 transgenic plants, 41 lines exhibited mutations and produced heritable TaSal1 mutations in the M1 progeny and 5 lines were full 5 gene knock-outs. These mutant plants exhibit a rolled-leaf phenotype in young leaves and bended stems, but there were no significant changes in the internode length and width, leaf morphology, and stem shape. Anatomical and scanning electron microscope studies of the young leaves of mutated TaSal1 lines showed closed stomata, increased stomata width and increase in the size of the bulliform cells. Sal1 mutant seedlings germinated and grew better on media containing polyethylene glycol than wildtype seedlings. Our results indicate that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing is efficient tool for mutating more multiple TaSal1 loci in hexaploid wheat.
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Recent advances in nanocomposite-based delivery systems for targeted CRISPR/Cas delivery and therapeutic genetic manipulation.
CRISPR/Cas systems are novel gene editing tools with tremendous capacity and accuracy for gene editing and hold great potential for therapeutic genetic manipulation. However, the lack of safe and efficient delivery methods for CRISPR/Cas and its guide RNA hinders their wide adoption for therapeutic applications. To this end, there is an increasing demand for safe, efficient, precise, and non-pathogenic delivery approaches, both in vitro and in vivo. With the convergence of nanotechnology and biomedicine, functional nanocomposites have demonstrated unparalleled sophistication to overcome the limits of CRISPR/Cas delivery. The tunability of the physicochemical properties of nanocomposites makes it very easy to conjugate them with different functional substances. The combinatorial application of diverse functional materials in the form of nanocomposites has shown excellent properties for CRISPR/Cas delivery at the target site with therapeutic potential. The recent highlights of selective organ targeting and phase I clinical trials for gene manipulation by CRISPR/Cas after delivery through LNPs are at the brink of making it to routine clinical practice. Here we summarize the recent advances in delivering CRISPR/Cas systems through nanocomposites for targeted delivery and therapeutic genome editing.
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Deep Learning Based Models for CRISPR/Cas Off-Target Prediction.
CRISPR/Cas genome editing technologies enable effective and controlled genetic modifications; however, off-target effects remain a significant concern, particularly in clinical applications. Experimental and in silico methods are developed to predict potential off-target sites (OTS), including deep learning based methods, which can automatically and comprehensively learn sequence features, offer a promising tool for OTS prediction. Here, this work reviews the existing OTS prediction tools with an emphasis on deep learning methods, characterizes datasets used for deep learning training and testing, and evaluates six deep learning models -CRISPR-Net, CRISPR-IP, R-CRISPR, CRISPR-M, CrisprDNT, and Crispr-SGRU -using six public datasets and validates OTS data from the CRISPRoffT database. Performance of these models is assessed using standardized metrics, such as Precision, Recall, F1 score, MCC, AUROC and PRAUC. This work finds that incorporating validated OTS datasets into model training enhanced overall model performance, and improved robustness of prediction, particularly with highly imbalanced datasets. While no model consistently outperforms other models across all scenarios, CRISPR-Net, R-CRISPR, and Crispr-SGRU show strong overall performance. This analysis demonstrates the importance of integrating high-quality validated OTS data with advanced deep learning architectures to improve CRISPR/Cas off-target site predictions, ensuring safer genome editing applications.
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Epigenome editing for targeted DNA (de)methylation: a new perspective in modulating gene expression.
Traditionally, it has been believed that inheritance is driven as phenotypic variations resulting from changes in DNA sequence. However, this paradigm has been challenged and redefined in the contemporary era of epigenetics. The changes in DNA methylation, histone modification, non-coding RNA biogenesis, and chromatin remodeling play crucial roles in genomic functions and regulation of gene expression. More importantly, some of these changes are inherited to the next generations as a part of epigenetic memory and play significant roles in gene expression. The sum total of all changes in DNA bases, histone proteins, and ncRNA biogenesis constitutes the epigenome. Continuous progress in deciphering epigenetic regulations and the existence of heritable epigenetic/epiallelic variations associated with trait of interest enables to deploy epigenome editing tools to modulate gene expression. DNA methylation marks can be utilized in epigenome editing for the manipulation of gene expression. Initially, genome/epigenome editing technologies relied on zinc-finger protein or transcriptional activator-like effector protein. However, the discovery of clustered regulatory interspaced short palindromic repeats CRISPR)/deadCRISPR-associated protein 9 (dCas9) enabled epigenome editing to be more specific/efficient for targeted DNA (de)methylation. One of the major concerns has been the off-target effects, wherein epigenome editing may unintentionally modify gene/regulatory element which may cause unintended change/harmful effects. Moreover, epigenome editing of germline cell raises several ethical/safety issues. This review focuses on the recent developments in epigenome editing tools/techniques, technological limitations, and future perspectives of this emerging technology in therapeutics for human diseases as well as plant improvement to achieve sustainable developmental goals.
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Interrogating Mitochondrial Biology and Disease Using CRISPR/Cas9 Gene Editing.
Mitochondrial disease originates from genetic changes that impact human bodily functions by disrupting the mitochondrial oxidative phosphorylation system. MitoCarta is a curated and published inventory that sheds light on the mitochondrial proteome, but the function of some mitochondrially-localised proteins remains poorly characterised. Consequently, various gene editing systems have been employed to uncover the involvement of these proteins in mitochondrial biology and disease. CRISPR/Cas9 is an efficient, versatile, and highly accurate genome editing tool that was first introduced over a decade ago and has since become an indispensable tool for targeted genetic manipulation in biological research. The broad spectrum of CRISPR/Cas9 applications serves as an attractive and tractable system to study genes and pathways that are essential for the regulation and maintenance of mitochondrial health. It has opened possibilities of generating reliable cell and animal models of human disease, and with further exploitation of the technology, large-scale genomic screenings have uncovered a wealth of fundamental mechanistic insights. In this review, we describe the applications of CRISPR/Cas9 system as a genome editing tool to uncover new insights into pathomechanisms of mitochondrial diseases and/or biological processes involved in mitochondrial function.
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Generation of albino medaka (Oryzias latipes) by CRISPR/Cas9.
CRISPR/Cas9 system is a powerful tool to produce the genetic modification in plants and animals such as mouse and zebrafish. However, this technique was less reported in fish model medaka (Oryzias latipes). Here, we describe an efficient and rapid procedure for genome editing in medaka tyr and generate a stable albino strain. The Cas9 mRNA and gRNA for tyr gene were injected into the embryos of orange-red medaka, and the tyr gene was disrupted in more than 90% of embryos in F0 and F1, which were validated by observation and sequencing of targeted locus. The pigment cells were largely decreased in the mutant medaka because open reading frames of tyr were shifted near the targeted locus, generating albino medaka. Taken together, this method provides a detailed procedure to generate the genetic modification medaka by using an optimized CRISPR/Cas9 system, and the new albino medaka provides an important research platform to study the pigmentation.
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