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Heritable gene editing in tomato through viral delivery of isopentenyl transferase and single-guide RNAs to latent axillary meristematic cells.
Realizing the full potential of genome editing for crop improvement has been slow due to inefficient methods for reagent delivery and the reliance on tissue culture for creating gene-edited plants. RNA viral vectors offer an alternative approach for delivering gene engineering reagents and bypassing the tissue culture requirement. Viruses, however, are often excluded from the shoot apical meristem, making virus-mediated gene editing inefficient in some species. Here, we developed effective approaches for generating gene-edited shoots in Cas9-expressing transgenic tomato plants using RNA virus-mediated delivery of single-guide RNAs (sgRNAs). RNA viral vectors expressing sgRNAs were either delivered to leaves or sites near axillary meristems. Trimming of the apical and axillary meristems induced new shoots to form from edited somatic cells. To further encourage the induction of shoots, we used RNA viral vectors to deliver sgRNAs along with the cytokinin biosynthesis gene, isopentenyl transferase. Abundant, phenotypically normal, gene-edited shoots were induced per infected plant with single and multiplexed gene edits fixed in the germline. The use of viruses to deliver both gene editing reagents and developmental regulators overcomes the bottleneck in applying virus-induced gene editing to dicotyledonous crops such as tomato and reduces the dependency on tissue culture.
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CRISPR/Cas9-mediated silencing of CD44: unveiling the role of hyaluronic acid-mediated interactions in cancer drug resistance.
A comprehensive overview of CD44 (CD44 Molecule (Indian Blood Group)), a cell surface glycoprotein, and its interaction with hyaluronic acid (HA) in drug resistance mechanisms across various types of cancer is provided, where CRISPR/Cas9 gene editing was utilized to silence CD44 expression and examine its impact on cancer cell behavior, migration, invasion, proliferation, and drug sensitivity. The significance of the HA-CD44 axis in tumor microenvironment (TME) delivery and its implications in specific cancer types, the influence of CD44 variants and the KHDRBS3 (KH RNA Binding Domain Containing, Signal Transduction Associated 3) gene on cancer progression and drug resistance, and the potential of targeting HA-mediated pathways using CRISPR/Cas9 gene editing technology to overcome drug resistance in cancer were also highlighted.
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Technical limitations of CRISPR-Cas9 genome editing in bacteria: challenges and future directions.
The CRISPR-Cas system, which originated as an adaptive immune system in bacteria and archaea, has been repurposed as a precise and programmable tool for genetic manipulation in both prokaryotes and eukaryotes. Its applications in bacteria include targeted genome modifications, antimicrobial resistance studies, functional genomics studies, and the development of engineered strains for industrial and synthetic biology applications. Therefore, the understanding of technical aspects of CRISPR systems and their underlying molecular mechanisms is essential for experimental accuracy, reproducibility, and biosafety. CRISPR editing introduces several challenges in bacteria, including off-target effects, DNA repair limitations, cytotoxicity of Cas nucleases, and host-specific restriction-modification barriers. In addition to these specific challenges, metabolic burden, sgRNA design, and delivery challenges further introduce limitations. Such issues compromise editing efficiency, genomic stability, and cell viability. Recent studies have focused on improved guide RNA design, alternative Cas variants, refined delivery strategies, and host-adapted engineering as promising directions to enhance editing. This review discusses the principal barriers to CRISPR-Cas9 genome editing of bacteria, evaluates the current strategies for addressing these barriers, and highlights emerging approaches aimed at improving the efficiency, reliability and precision in bacterial genome engineering.
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Inhibition of histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing.
Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1-9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.
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Advanced gene editing techniques for enhancing disease resistance and climate resilience in crops.
Ensuring food security and solving the issues brought on by climate change require breeding and engineering of climate-resilient crops. Despite its contributions to reducing agricultural diseases, genetic engineering has several limitations, including high labor costs, lengthy processing times, and poor productivity. Genome editing has become a potential method to provide notable opportunities to explain complex biological processes, genetically solve the causes of diseases, and improve crops for disease resistance by effectively modifying multiple traits. Genome editing techniques including TALENs, ZFNs, and CRISPR/Cas9 increase agricultural productivity by developing climate-resistant crops and promoting climate-resilient agriculture. Among these approaches, CRISPR/Cas9 shows exceptional efficacy, minimal chance of off-target effects, and improved traits such as drought tolerance and disease resistance. This study explores advanced gene editing techniques for improving disease resistance in crops and developing climate-resilient varieties to reduce food insecurity and hunger. It demonstrates that these techniques have enhanced the nutritional content and resilience of many crops by fighting abiotic and biotic stresses. Future agricultural practices could alter the genes and improve disease-resistant crops by genome editing techniques.
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CRISPR/Cas: from adaptive immune system in prokaryotes to therapeutic weapon against immune-related diseases.
CRISPR/Cas evolved as an adaptive immune system in bacteria and archaea to inactivate foreign viral and plasmid DNA. However, the capacities of various CRISPR/Cas systems for precise genome editing based on sequence homology also allow their use as tools for genomic and epigenomic modification in eukaryotes. Indeed, these genetic characteristics have proven useful for disease modeling and testing the specific functions of target genes under pathological conditions. Moreover, recent studies provide compelling evidence that CRISPR/Cas systems could be useful therapeutic tools against human diseases, including cancer, monogenic disorders, and autoimmune disorders.HighlightsCRISPR/Cas evolved as an adaptive immune system in bacteria and archaea.CRISPR/Cas systems are nowadays used as tools for genomic modification.CRISPR/Cas systems could be useful therapeutic tools against human disease, including autoimmune conditions.
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The illusion of control in germline-engineering policy.
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High-Frequency Generation of Homozygous/Biallelic Mutants via CRISPR/Cas9 Driven by AtKu70/80 Promoters.
CRISPR/Cas9 gene editing technology is widely used in plant gene editing to verify gene function or improve agronomic traits. In the CRISPR/Cas9 system, Cas9 expression hinges on promoter choice, and CRISPR/Cas9 driven by a strong promoter or cell division-specific promoter has a higher editing efficiency. The CRISPR/Cas9 mechanism involves the CAS9 enzyme, which, directed by guide RNA, cleaves target double-stranded DNA and subsequently induces insertions or deletions (InDels) through the non-homologous end joining (NHEJ) repair pathway. The Ku protein plays a central role in the NHEJ repair process. It remains unclear whether driving Cas9 with promoters of AtKu70 and AtKu80, which are subunits of the Ku protein, will enhance gene editing efficiency. In this study, the promoters of AtKu70 and AtKu80 were cloned and used to drive Cas9 in the CRISPR/Cas9 system. Four different genes, GmRj7, GmNNL1, AtPDS3, and AtBRI1, were designed for soybean hairy root transformation and Arabidopsis transformation. The results showed that the CRISPR/Cas9 systems driven by the promoters of AtKu70 and AtKu80 achieved higher homozygous/biallelic mutation efficiencies than the CRISPR/Cas9 system driven by the 35S promoter in hairy root transformation by Rhizobium rhizogenes and stable genetic transformation with Rhizobium tumefaciens.
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CRISPR-Cas9-Mediated Gene Editing in Mouse Spermatogonial Stem Cells.
Precise genome editing is a powerful tool for analysis of gene function. However, in spermatogonial stem cells (SSCs), this still remains a big challenge mainly due to low efficiency and complexity of currently available gene editing techniques. The CRISPR-Cas9 system from bacteria has been applied to modifying genome in different species at a very high efficiency and specificity. Here we describe CRISPR-Cas9-mediated gene editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in SSCs. This protocol provides guidelines for derivation of SSCs, nucleofection of SSCs with the CRISPR-Cas9 system, transplantation of the gene-modified SSCs into the recipient testes, and production of mice using transplanted SSC-derived round spermatids.
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CRISPR-Cas9 Targeting PCSK9: A Promising Therapeutic Approach for Atherosclerosis.
CRISPR-Cas9 gene editing technology, as an innovative biomedical tool, holds significant potential in the prevention and treatment of atherosclerosis. By precisely editing key genes such as PCSK9, CRISPR-Cas9 offers the possibility of long-term regulation of low-density lipoprotein cholesterol (LDL-C), which may reduce the risk of cardiovascular diseases. Early clinical studies of gene editing therapies like VERVE-101 have yielded encouraging results, highlighting both the feasibility and potential efficacy of this technology. However, clinical applications still face challenges such as off-target effects, immunogenicity, and long-term safety. Future research should focus on enhancing the specificity and efficiency of gene editing, optimizing delivery systems, and improving personalized treatment strategies. Additionally, the establishment of ethical and legal regulatory frameworks will be critical for the safe adoption of this technology. With the continued advancement of gene editing technology, CRISPR-Cas9 may become an important tool for treating atherosclerosis and other complex diseases.
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