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KEAP1 Editing Using CRISPR/Cas9 for Therapeutic NRF2 Activation in Primary Human T Lymphocytes.
Oxidant stress modifies T lymphocyte activation and function. Previous work demonstrated that murine T cell-specific kelch like-ECH-associated protein 1 (Keap1) deletion enhances antioxidant capacity and protects from experimental acute kidney injury. In this study, we used CRISPR technology to develop clinically translatable human T cell-specific KEAP1 deletion. Delivery of KEAP1 exon 2 specific Cas9:guide RNA in Jurkat T cells led to significant (∼70%) editing and upregulation of NRF2-regulated antioxidant genes NADPH dehydrogenase quinone 1 (NQO1) (up to 11-fold), heme oxygenase 1 (HO1) (up to 11-fold), and GCLM (up to 2-fold). In primary human T cells, delivery of KEAP1 exon 2 target site 2-specific ATTO 550-labeled Cas9:guide RNA edited KEAP1 in ∼40% cells and significantly (p ≤ 0.04) increased NQO1 (16-fold), HO1 (9-fold), and GCLM (2-fold) expression. To further enrich KEAP1-edited cells, ATTO 550-positive cells were sorted 24 h after electroporation. Assessment of ATTO 550-positive cells showed KEAP1 editing in ∼55% cells. There was no detectable off-target cleavage in the top three predicted genes in the ATTO 550-positive cells. Gene expression analysis found significantly (p ≤ 0.01) higher expression of NQO1 mRNA in ATTO 550-positive cells compared with control cells. Flow cytometric assessment showed increased (p ≤ 0.01) frequency of CD4-, CD25-, and CD69-expressing KEAP1 edited cells whereas frequency of CD8- (p ≤ 0.01) and IL-17- (p ≤ 0.05) expressing cells was reduced compared with control cells. Similar experimental conditions resulted in significant KEAP1 editing, increased antioxidant gene expression, and frequency of CD69 and IL-10 positive cells in highly enriched KEAP1-edited regulatory T cells. KEAP1-edited T cells could potentially be used for treating multiple human diseases.
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CRISPR as a therapeutic tool for inherited retinal degenerations: Advances, challenges, and future directions.
Inherited retinal diseases (IRDs) are a genetically diverse group of disorders characterized by progressive photoreceptor degeneration, leading to vision loss and blindness. With over 320 associated genes and significant phenotypic variability, effective treatment remains challenging. Recent advances in genome editing, particularly CRISPR/Cas-based technologies, have revolutionized therapeutic approaches by enabling precise and customizable DNA and RNA editing. This review explores the application of various CRISPR strategies-such as gene knockout via non-homologous end joining (NHEJ), exon skipping using dual-sgRNAs, homology-directed repair (HDR), base editing (BE), prime editing (PE), RNA editing with Cas13, and epigenetic modulation through CRISPRa/i-in preclinical models of IRDs. Emphasis is placed on allele-specific targeting, gene-agnostic approaches, and mutation-independent strategies to address dominant and recessive forms of disease. We also highlight recent clinical milestones, including the first human trial using CRISPR gene editing for CEP290-associated Leber congenital amaurosis. Finally, we discuss critical challenges, including delivery constraints, immune responses, and off-target effects, along with emerging solutions such as engineered Cas variants, split-intein systems, and advanced off-target detection methods. Together, these advances underscore the transformative potential of CRISPR technologies in treating IRDs and lay the foundation for future clinical translation.
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High-Throughput Base Editing-Mediated Artificial Evolution Streamlines Trait Gene Identification in Rice.
Base Editing-Mediated Gene Evolution (BEMGE) method develops novel rice germplasms with mutations in any endogenous gene through employing both Cas9n-based cytosine and adenine base editors combined with an sgRNA library tiling the full-length coding region, for obtaining unknown functional SNPs. Here we describe the process of artificial evolution of OsALS1 in rice cells using BEMGE through Agrobacterium-mediated transformation. BEMGE method has the potential to generate numerous nucleotide changes to screen pivotal amino acids (AAs) and to accelerate crop improvement.
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Advances in CRISPR Base Editing: From Molecular Evolution to Therapeutic Applications in Genomic Medicine.
CRISPR-Cas9 systems revolutionized gene editing, but inherent drawbacks, namely DNA double-strand breaks (DSBs) and the difficulty of achieving precise repairs (due to low HDR efficiency), led researchers to invent new, more accurate gene editing tools. Base editing represents a significant leap forward, enabling targeted single-nucleotide conversions directly on the DNA without DSBs or donor templates. The core technology involves fusing catalytically dead or nickase Cas proteins to DNA deaminase enzymes. Cytosine base editors (CBEs) convert C•G to T•A pairs, while adenine base editors (ABEs) change A•T to G•C. These editors exploit the deaminase function within the R-loop structure formed by Cas binding and co-opt endogenous DNA repair mechanisms for precision. While offering improved efficiency and editing precision, base editing faces persistent challenges, such as off-target effects, bystander edits, delivery and ethical concerns. Continuous engineering efforts have refined these tools, enhancing accuracy, expanding targetability and reducing unwanted edits. The base editing arsenal has also broadened to include C-to-G base editors (CGBEs), dual A&C editors and versions targeting organelles. Successful preclinical studies demonstrating the correction of mutations responsible for the disease have paved the way for clinical trials, which are now testing therapies for conditions like sickle cell disease, β-thalassaemia and hypercholesterolemia using various delivery systems. This review explores CRISPR base editing's origins, mechanisms of action, potential therapies and current restrictions, pointing to its broadening impact on medical genetics.
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APOBEC: From mutator to editor.
APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) are a family of cytidine deaminases that prefer single-stranded nucleic acids as substrates. Besides their physiological functions, APOBEC family members have been found to cause hypermutations of cancer genomes, which could be correlated with cancer development and poor prognosis. Recently, APOBEC family members have been combined with the versatile CRISPR/Cas9 system to perform targeted base editing or induce hypermutagenesis. This combination improved the CRISPR/Cas9-mediated gene editing at single-base precision, greatly enhancing its usefulness. Here, we review the physiological functions and structural characteristics of APOBEC family members and their roles as endogenous mutators that contribute to hypermutations during carcinogenesis. We also review the various iterations of the APOBEC-CRISPR/Cas9 gene-editing tools, pointing out their features and limitations as well as the possibilities for future developments.
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FAME-CRISPR improves CRISPR-Cas9 genome editing via HDAC inhibition and engineered virus-like particle delivery.
CRISPR-mediated gene editing using engineered virus-like particles (eVLPs) can achieve high efficiency, but performance varies with reduced effectiveness often seen in primary cells or when generating polyclonal models at scale. We developed a faster, accurate and 4-fold more efficient CRISPR-Cas9 (FAME-CRISPR) method using pan-histone deacetylase inhibitors with eVLP transduction compared to previous reports using other histone deacetylase inhibitors. Combined optimization of pan-HDACi treatment with eVLP enhanced double-strand break (DSB)-mediated CRISPR and base editing gave significantly edited populations within 2- to 3-cell mean population doublings, reducing the need for post-editing selection in immortalized cancer cells and in primary diploid fibroblasts that have limited replicative lifespans.
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Advancements of CRISPR-Mediated Base Editing in Crops and Potential Applications in Populus.
Base editing represents a cutting-edge genome editing technique that utilizes the CRISPR system to guide base deaminases with high precision to specific genomic sites, facilitating the targeted alteration of individual nucleotides. Unlike traditional gene editing approaches, base editing does not require DNA double-strand breaks or donor templates. It functions independently of the cellular DNA repair machinery, offering significant advantages in terms of both efficiency and accuracy. In this review, we summarize the core design principles of various DNA base editors, their distinctive editing characteristics, and tactics to refine their efficacy. We also summarize their applications in crop genetic improvement and explore their potential contributions to forest genetic engineering.
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Emerging applications of gene editing technologies for the development of climate-resilient crops.
Climate change threatens global crop yield and food security due to rising temperatures, erratic rainfall, and increased abiotic stresses like drought, heat, and salinity. Gene editing technologies, including CRISPR/Cas9, base editors, and prime editors, offer precise tools for enhancing crop resilience. This review explores the mechanisms of these technologies and their applications in developing climate-resilient crops to address future challenges. While CRISPR/enables targeted modifications of plant DNA, the base editors allow for direct base conversion without inducing double-stranded breaks, and the prime editors enable precise insertions, deletions, and substitutions. By understanding and manipulating key regulator genes involved in stress responses, such as DREB, HSP, SOS, ERECTA, HsfA1, and NHX; crop tolerance can be enhanced against drought, heat, and salt stress. Gene editing can improve traits related to root development, water use efficiency, stress response pathways, heat shock response, photosynthesis, membrane stability, ion homeostasis, osmotic adjustment, and oxidative stress response. Advancements in gene editing technologies, integration with genomics, phenomics, artificial intelligence (AI)/machine learning (ML) hold great promise. However, challenges such as off-target effects, delivery methods, and regulatory barriers must be addressed. This review highlights the potential of gene editing to develop climate-resilient crops, contributing to food security and sustainable agriculture.
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Plant base editing and prime editing: The current status and future perspectives.
Precise replacement of an allele with an elite allele controlling an important agronomic trait in a predefined manner by gene editing technologies is highly desirable in crop improvement. Base editing and prime editing are two newly developed precision gene editing systems which can introduce the substitution of a single base and install the desired short indels to the target loci in the absence of double-strand breaks and donor repair templates, respectively. Since their discoveries, various strategies have been attempted to optimize both base editor (BE) and prime editor (PE) in order to improve the precise editing efficacy, specificity, and expand the targeting scopes. Here, we summarize the latest development of various BEs and PEs, as well as their applications in plants. Based on these progresses, we recommend the appropriate BEs and PEs for both basic plant research and crop improvement. Moreover, we propose the perspectives for further optimization of these two editors. We envision that both BEs and PEs will become the routine and customized precise gene editing tools for both plant biological research and crop improvement in the near future.
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Genomic and biochemical comparison of allelic triple-mutant lines derived from conventional breeding and multiplex gene editing.
Multiplex gene editing allows for the simultaneous targeting and mutagenesis of multiple loci in a genome. This tool is particularly valuable for plant genetic improvement, as plant genomes often require mutations at multiple loci to confer useful and/or novel traits. However, the regulation of gene editing can vary depending on the number of loci targeted. In this study, we developed triple-mutant soybean (Glycine max (L.) Merrill) lines using different crop improvement strategies, including conventional backcross breeding of standing variant alleles and clustered regularly interspaced short palindromic repeats-based multiplex editing to introduce new alleles. The mutations were targeted to genes encoding seed antinutritional components, as previously described in a triple null soybean carrying knockout alleles for a Kunitz trypsin inhibitor, a soybean agglutinin, and the allergen P34 protein. The products developed from these respective genetic improvement pipelines were tested for differences between the triple-mutant lines and their parental lines. Analyses included genomics, seed proteomics, trypsin inhibition, seed protein digestibility, and harvestable yield of the different lines. We observed that both multiplex gene editing and conventional breeding approaches produced essentially equivalent products in comparison to their parental lines. We conclude that the multiplex gene editing strategy is not inherently riskier than conventional breeding for developing complex mutant lines of this type.
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