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CRISPRthripsis: The Risk of CRISPR/Cas9-induced Chromothripsis in Gene Therapy.
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system has allowed the generation of disease models and the development of therapeutic approaches for many genetic and non-genetic disorders. However, the generation of large genomic rearrangements has raised safety concerns for the clinical application of CRISPR/Cas9 nuclease approaches. Among these events, the formation of micronuclei and chromosome bridges due to chromosomal truncations can lead to massive genomic rearrangements localized to one or few chromosomes. This phenomenon, known as chromothripsis, was originally described in cancer cells, where it is believed to be caused by defective chromosome segregation during mitosis or DNA double-strand breaks. Here, we will discuss the factors influencing CRISPR/Cas9-induced chromothripsis, hereafter termed CRISPRthripsis, and its outcomes, the tools to characterize these events and strategies to minimize them.
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Trichostatin A for Efficient CRISPR-Cas9 Gene Editing of Human Pluripotent Stem Cells.
Genome-edited human-induced pluripotent stem cells (iPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. Despite the development of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, the gene editing process is inefficient and can take several weeks to months to generate edited iPSC clones. We developed a strategy to improve the efficiency of the iPSC gene editing process via application of a small-molecule, trichostatin A (TSA), a Class I and II histone deacetylase inhibitor. We observed that TSA decreased global chromatin condensation and further resulted in increased gene-editing efficiency of iPSCs by twofold to fourfold while concurrently ensuring no increased off-target effects. The edited iPSCs could be clonally expanded while maintaining genomic integrity and pluripotency. The rapid generation of therapeutically relevant gene-edited iPSCs could be enabled by these findings.
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CRISPR Screening in Hepatocellular Carcinoma: From Tumor Progression to Immune Evasion and Therapeutic Resistance.
Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and a leading cause of cancer-related mortality worldwide. Despite advances in targeted therapies and immunotherapies, clinical outcomes remain poor owing to profound molecular heterogeneity, intrinsic therapeutic resistance, and complex immune evasion mechanisms. Although genomic profiling has identified recurrent alterations in HCC, large-scale functional validation of candidate drivers and vulnerabilities remains challenging. CRISPR (clustered regularly interspaced short palindromic repeats)-based screening technologies have transformed this landscape by enabling systematic interrogation of gene function in physiologically relevant contexts. In this review, we summarize recent studies that have applied CRISPR screening approaches in HCC research. These efforts have uncovered multilayered dependency programs that govern ferroptosis resistance, metabolic reprogramming, epigenetic regulation, tumor suppressor networks, immune evasion, and resistance to targeted therapies. We also discuss the major limitations of current studies, including model bias, incomplete representation of HCC heterogeneity, and technical constraints intrinsic to pooled screening. Overall, integration of CRISPR screening with patient-derived models, single-cell readouts, and precision editing technologies is expected to accelerate mechanistic discovery and biomarker-guided therapeutic prioritization for HCC.
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Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.
The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.
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CRISPR-Cas technology based genome editing for modification of salinity stress tolerance responses in rice (Oryza sativa L.).
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas) technology is an effective tool for site-specific genome editing, used to precisely induce mutagenesis in different plant species including rice. Salinity is one of the most stressful environmental constraints affecting agricultural productivity worldwide. As plant adaptation to salinity stress is under polygenic control therefore, 51 rice genes have been identified that play crucial role in response to salinity. This review offers an exclusive overview of genes identified in rice genome for salinity stress tolerance. This will provide an idea to produce rice varieties with enhanced salt tolerance using the potentially efficient CRISPR-Cas technology. Several undesirable off-target effects of CRISPR-Cas technology and their possible solutions have also been highlighted.
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Design, execution, and analysis of pooled in vitro CRISPR/Cas9 screens.
The recently described clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has proven to be an exquisitely powerful and invaluable method of genetic manipulation and/or modification. As such, many researchers have realized the potential of using the CRISPR/Cas9 system as a novel screening method for the identification of important proteins in biological processes and have designed short guide RNA libraries for an in vitro screening. The seminal papers describing these libraries offer valuable information regarding methods for generating the short guide RNA libraries, creating cell lines containing these libraries, and specific details regarding the screening workflow. However, certain considerations are often overlooked that may be important when planning and performing a screen, including which CRISPR library to use and how to best analyze the resulting screen data. In this review, we offer suggestions to answer some of these questions that are not covered as deeply in the papers describing the available CRISPR libraries for an in vitro screening.
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Genome editing in cardiovascular diseases.
Genetic modification at the molecular level in somatic cells, germline, and animal models requires for different purposes, such as introducing desired mutation, deletion of alleles, and insertion of novel genes in the genome. Various genome-editing tools are available to accomplish these alterations, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) system. CRISPR-Cas system is an emerging technology, which is being used in biological and medical sciences, including in the cardiovascular field. It assists to identify the mechanism of various cardiovascular disease occurrence, such as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic cardiomyopathy (ACM). Furthermore, it has been advantages to edit various genes simultaneously and can also be used to treat and prevent several human diseases. This chapter explores the use of the scientific and therapeutic potential of a CRISPR-Cas system to edit the various cardiovascular disease-associated genes to understand the pathways involved in disease progression and treatment.
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Recent advances in the delivery and applications of nonviral CRISPR/Cas9 gene editing.
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system has been a major technological breakthrough that has brought revolutionary changes to genome editing for therapeutic and diagnostic purposes and precision medicine. With the advent of the CRISPR/Cas9 system, one of the critical limiting factors has been the safe and efficient delivery of this system to cells or tissues of interest. Several approaches have been investigated to find delivery systems that can attain tissue-targeted delivery, lowering the chances of off-target editing. While viral vectors have shown promise for in vitro, in vivo and ex vivo delivery of CRISPR/Cas9, their further clinical applications have been restricted due to shortcomings including limited cargo packaging capacity, difficulties with large-scale production, immunogenicity and insertional mutagenesis. Rapid progress in nonviral delivery vectors, including the use of lipid, polymer, peptides, and inorganic nanoparticle-based delivery systems, has established nonviral delivery approaches as a viable alternative to viral vectors. This review will introduce the molecular mechanisms of the CRISPR/Cas9 gene editing system, current strategies for delivering CRISPR/Cas9-based tools, an overview of strategies for overcoming off-target genome editing, and approaches for improving genome targeting and tissue targeting. We will also highlight current developments and recent clinical trials for the delivery of CRISPR/Cas9. Finally, future directions for overcoming the limitations and adaptation of this technology for clinical trials will be discussed.
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Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing.
RATIONALE: Individuals with naturally occurring loss-of-function proprotein convertase subtilisin/kexin type 9 (PCSK9) mutations experience reduced low-density lipoprotein cholesterol levels and protection against cardiovascular disease. OBJECTIVE: The goal of this study was to assess whether genome editing using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system can efficiently introduce loss-of-function mutations into the endogenous PCSK9 gene in vivo. METHODS AND RESULTS: We used adenovirus to express CRISPR-associated 9 and a CRISPR guide RNA targeting Pcsk9 in mouse liver, where the gene is specifically expressed. We found that <3 to 4 days of administration of the virus, the mutagenesis rate of Pcsk9 in the liver was as high as >50%. This resulted in decreased plasma PCSK9 levels, increased hepatic low-density lipoprotein receptor levels, and decreased plasma cholesterol levels (by 35-40%). No off-target mutagenesis was detected in 10 selected sites. CONCLUSIONS: Genome editing with the CRISPR-CRISPR-associated 9 system disrupts the Pcsk9 gene in vivo with high efficiency and reduces blood cholesterol levels in mice. This approach may have therapeutic potential for the prevention of cardiovascular disease in humans.
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Tailoring Bacterial Cellulose through the CRISPR/Cas9-Mediated Gene Editing Tool in Komagataeibacter xylinus.
Bacterial cellulose (BC) is a nanocellulose produced by bacteria, formed by glucose units linked through β-1,4 glycosidic bonds. It features a three-dimensional network structure, superior water retention capacity, high porosity, and outstanding biocompatibility, among other notable characteristics. Komagataeibacter xylinus was the predominant strain used for BC production. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associate-protein 9)-mediated gene editing tool has been applied in various species; however, its application in K. xylinus has not been reported. To facilitate metabolic pathway engineering in K. xylinus, a CRISPR/Cas9-mediated gene editing tool specific to this strain was developed, achieving a gene editing efficiency exceeding 73%. Upon application of the CRISPR/Cas9-mediated gene editing tool in K. xylinus, the strain's ability to synthesize BC was enhanced by 23.6% (5.75 g/L), and the impact of BC synthase-correlated genes (bcsH, bcsX, bcsY, and bcsZ) on BC structure was investigated. The advancement of CRISPR/Cas9-mediated gene editing tools in K. xylinus is expected to accelerate genetic modification of this organism. This advancement has the potential to significantly improve our understanding of the genetic regulatory mechanisms that govern the structure and production of BC, thereby facilitating cost-effective synthesis of BC with tailored structural properties.
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