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Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors.
Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy.
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CRISPR/Cas-based genome editing for enhancing grain quality traits in rice (Oryza sativa L.).
Rice is one of the most important food crops and feeds more than half of the world's population. Enhancing grain quality is currently a highly important issue since consumers are now more concerned with the taste, appearance, and nutritional value of the grain. The quality of grain in rice is complex and regulated by a multitude of genes that influence qualities such as amylase content, grain size and shape, chalkiness, aroma and nutrient content. The traditional forms of breeding, such as hybridization and marker-assisted selection, are slow and less effective since such characteristics are regulated by many genes and are influenced by environmental conditions. CRISPR/Cas genome editing has become a potent tool that enables scientists to directly and specifically edit grain quality-related genes. Important genes such as Wx (amylose), GS3, GW8, and TGW3 (grain size), Chalk5 (chalkiness), BADH2 (aroma), and nutrient-related grain size genes such as OsAAP6, OsAAP10, and OsVIT1/2, have been successfully edited to enhance the quality of rice. Newer methods, such as base editing and prime editing, enable this process to become even more precise by modifying the specific bases of DNA without cutting the DNA. CRISPR has assisted in the improvement of rice by controlling the amylose content, reducing chalkiness, enhancing aroma, and improving nutritional quality. It is more accurate and quicker than traditional breeding, and it can enhance various traits simultaneously. Nonetheless, other challenges, such as off-target effects, regulatory concerns, and acceptance by the people, still have to be overcome. Combining CRISPR with artificial intelligence and genomic selection in the future will aid in creating superior versions of rice in shorter periods of time. Overall, CRISPR/Cas genome editing is a potential method to enhance the quality of rice and secure food security in the whole world.
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Megabase-scale loss of heterozygosity provoked by CRISPR-Cas9 DNA double-strand breaks.
Harnessing DNA double-strand breaks (DSBs) is a powerful approach for gene editing, but it may provoke loss of heterozygosity (LOH), a common feature of tumor genomes. To interrogate this risk, we developed a flow cytometry-based system (Flo-LOH), detecting LOH in ∼5% of mouse embryonic and human epithelial cells following a DSB. Inhibition of both non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) massively increases LOH, although the dependence on individual pathways differs in the two cell types. Multiple mechanisms lead to LOH, including chromosome truncations with de novo telomere addition and whole chromosome loss. LOH spans megabases distal from the DSB but also frequently tens of megabases centromere-proximal, which can arise from breakage-fusion-bridge events. Unlike DSBs, Cas9 nicks and adenine base editing did not noticeably impact LOH. The capacity for large-scale LOH must therefore be considered when using DSB-based gene editing, especially in conjunction with end-joining inhibition.
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Precision Transcriptome Editing.
Manipulating RNA species in mammalian cells has emerged as an important strategy for precise gene expression control. Here we review recent advances in precision transcriptome editing with a focus on tools that engineer specific transcripts for abundance, translation, base editing, alternative isoforms, and chemical modifications. While some of these methods have demonstrated efficiency in therapeutically relevant cellular or in vivo models, most require further study on their clinical safety and efficacy. Precision transcriptome engineering holds great potential for both mechanistic study of RNA biology and future gene and cell-based therapeutic applications.
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Restoring T and B cell generation in X-linked severe combined immunodeficiency mice through hematopoietic stem cells adenine base editing.
Base editing of hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematologic diseases. However, the feasibility of using adenine-base-edited HSPCs for treating X-linked severe combined immunodeficiency (SCID-X1), the influence of dose-response relationships on immune cell generation, and the potential risks have not been demonstrated in vivo. Here, a humanized SCID-X1 mouse model was established, and 86.67% ± 2.52% (n = 3) of mouse hematopoietic stem cell (HSC) pathogenic mutations were corrected, with no single-guide-RNA (sgRNA)-dependent off-target effects detected. Analysis of peripheral blood over 16 weeks post-transplantation in mice with different immunodeficiency backgrounds revealed efficient immune cell generation following transplantation of different amounts of modified HSCs. Therefore, a large-scale infusion of gene-corrected HSCs within a safe range can achieve rapid, stable, and durable immune cell regeneration. Tissue-section staining further demonstrated the restoration of immune organ tissue structures, with no tumor formation in multiple organs. Collectively, these data suggest that base-edited HSCs are a potential therapeutic approach for SCID-X1 and that a threshold infusion dose of gene-corrected cells is required for immune cell regeneration. This study lays a theoretical foundation for the clinical application of base-edited HSCs in treating SCID-X1.
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A streamlined base editor engineering strategy to reduce bystander editing.
Base editing (BE) can permanently correct over half of known human pathogenic genetic variants without requiring a repair template, thus serving as a promising therapeutic tool to treat a broad spectrum of genetic diseases. However, the broad activity windows of current base editors pose a major challenge to their therapeutic application. Here, we show that integrating a naturally occurring oligonucleotide binding module into the deaminase active center of TadA-8e, a highly active deoxyadenosine deaminase, enhances its editing specificity. When conjugated with a Cas9 nickase or alternative PAM Cas9 variants, the engineered TadA variant-TadA-NW1-consistently achieves robust A-to-G editing efficiencies within an editing window consisting of four nucleotides, substantially narrower than the 10-bp editing window of the TadA-8e-derived ABEs. Moreover, compared to ABE8e, ABE-NW1 shows significantly decreased Cas9-dependent and -independent off-target activity while maintaining similar on-target editing efficiency. Further, TadA-NW1 can be reprogrammed to perform desired cytidine deamination and adenine transversion within a restricted editing window. Finally, in a cystic fibrosis (CF) cell model, ABE-NW1 outperforms existing ABEs in accurately and efficiently correcting the CFTR W1282X variant, one of the most common CF-causing mutations. In all, we engineered a suite of base editors with refined activity windows, enabling more precise base editing. Importantly, this study presents a streamlined genome editor re-engineering strategy to accelerate the development of therapeutic base editing.
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Revolutionizing Heart Valve Therapy: A Translational Framework for Combining Decellularized Scaffolds With Genetic Modification.
Valvular heart disease (VHD) represents a significant global health challenge, affecting over 2.5% of the population and disproportionately impacting older adults due to age-related degenerative processes. Current treatment options-mechanical and bioprosthetic valves-both present substantial limitations that impact patient quality of life and long-term outcomes. Mechanical valves require lifelong anticoagulation with associated bleeding risks, while bioprosthetic valves suffer from limited durability, typically requiring replacement within 10-15 years. These limitations are particularly problematic for pediatric and young adult populations, who face multiple surgical interventions throughout their lifetime. Decellularized scaffolds have emerged as promising alternatives due to their natural extracellular matrix architecture and potential for recipient cell repopulation and growth, making them especially valuable for pediatric patients. However, these scaffolds face several critical biological challenges that have prevented widespread clinical adoption, including suboptimal recellularization patterns, inflammatory responses, calcification propensity, and structural degradation over time. Recent advances in gene editing technologies, particularly CRISPR-Cas9 and emerging base editing techniques, offer unprecedented opportunities to overcome these limitations by enabling precise molecular-level tissue modifications. These technologies could enhance scaffold biocompatibility, promote favorable cellular responses, prevent calcification, and improve structural integrity. This review explores how combining decellularized scaffold approaches with gene editing techniques could transform heart valve treatment. We examine the potential of this integrated approach to create valve replacements with improved durability and biocompatibility and propose pathways for translating these innovations into clinical practice. The convergence of these technologies holds particular promise for younger patients who would benefit most from heart valves capable of growth and long-term function without repeated surgical interventions.
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CRISPR Base Editing Correction of TGFBI Mutations in Autosomal Dominant Corneal Dystrophies.
PURPOSE: Lattice and granular corneal dystrophy comprise two common TGFBI-associated autosomal dominant corneal disorders. Existing therapies are only temporizing and carry significant morbidity. Here, we develop a novel therapeutic approach using an adenine base editor (ABE) to correct common TGFBI mutations. METHOD: We generated two human corneal epithelial (HCE) cell models harboring a copy of the most common disease-causing TGBFI mutations, R124C or R555W. These lines were electroporated with an ABE8e-NG-encoding mRNA and guide RNAs targeting the mutations. The resulting A•T-to-G•C editing efficiencies and off-target (OT) effects were assessed by amplicon sequencing. GFP-expressing adeno-associated viruses (AAVs) with different capsid types were transduced into HCE cells and healthy human corneal donor tissues, and GFP fluorescence was evaluated. RESULTS: Using all-RNA delivery for ABE8e-NG, we achieved 91% and 62% correction of the pathogenic adenines in HCE TGFBIR124C/WT and TGFBIR555W/WT cells, without editing the wild-type allele. Indel formation was negligible (<0.2%), bystander adenine editing was minimal (<0.7%), and editing at top computationally predicted OT sites was modest (<1.2% at all but 1 of the 20 OT sites analyzed), suggesting minimal safety concerns. Correction of TGFBIR124C/WT in HCEs rescued the aberrant lysosomal localization of TGFBI. We further identified AAV1 as the most effective serotype for gene delivery into both human corneal donor tissue and HCE cells. CONCLUSIONS: Our study demonstrates the feasibility and safety of CRISPR adenine base editing as a new therapeutic strategy for correcting common TGFBI mutations in corneal dystrophies, paving the way for further preclinical testing.
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CRISPR/Cas9-based nanodelivery systems for precision gene editing in neuroinflammation.
BACKGROUND: Neuroinflammation is a characteristic feature of many neurological diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Uncontrolled activation of microglia and astrocytes results in the production of proinflammatory mediators that contribute to neuronal dysfunction and degeneration. PURPOSE: CRISPR/Cas9 genome-editing technology provides highly precise targeting of genes involved in these pathways, enabling the deletion, correction, or modulation of disease-associated genetic alterations. Nonetheless, effective delivery of CRISPR components to the central nervous system (CNS) remains a major challenge because of protective barriers such as the blood-brain barrier (BBB), as well as concerns regarding genomic instability and immunogenicity. METHODS: Nanotechnology-guided delivery platforms, such as lipid nanoparticles, liposomes, dendrimers and polymeric carriers, have been developed as promising strategies to address these challenges. These nanosystems can improve biodistribution, pharmacokinetic behavior, and cellular uptake, while potentially reducing off-target effects and systemic toxicity. RESULTS: The preclinical studies demonstrate successful modulation of key neuroinflammatory targets, including the NLRP3 inflammasome, triggering receptor expressed on myeloid cells 2 (TREM2), Janus kinase (JAK) signaling, and the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, with potential therapeutic relevance to neurodegenerative diseases. In addition, novel techniques, such as mitochondrial base editing, prime editing, and epigenetic modulation, expand the scope of CRISPR-mediated therapeutic strategies. Although outcomes from animal models are promising, translation to the clinical setting requires careful consideration of safety, ethical concerns, and long-term efficacy. CONCLUSIONS: This review highlights the potential of integrating CRISPR/Cas9 with nanodelivery systems to advance precision medicine for neuroinflammatory disorders, while emphasizing their potential for therapeutic applications in complex CNS conditions. Ongoing studies aimed at optimizing delivery and reducing off-target effects will be essential for advancing these approaches toward clinical practice.
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Aberrant enhancer hypomethylation contributes to hepatic carcinogenesis through global transcriptional reprogramming.
Hepatocellular carcinomas (HCC) exhibit distinct promoter hypermethylation patterns, but the epigenetic regulation and function of transcriptional enhancers remain unclear. Here, our affinity- and bisulfite-based whole-genome sequencing analyses reveal global enhancer hypomethylation in human HCCs. Integrative epigenomic characterization further pinpoints a recurrent hypomethylated enhancer of CCAAT/enhancer-binding protein-beta (C/EBPβ) which correlates with C/EBPβ over-expression and poorer prognosis of patients. Demethylation of C/EBPβ enhancer reactivates a self-reinforcing enhancer-target loop via direct transcriptional up-regulation of enhancer RNA. Conversely, deletion of this enhancer via CRISPR/Cas9 reduces C/EBPβ expression and its genome-wide co-occupancy with BRD4 at H3K27ac-marked enhancers and super-enhancers, leading to drastic suppression of driver oncogenes and HCC tumorigenicity. Hepatitis B X protein transgenic mouse model of HCC recapitulates this paradigm, as C/ebpβ enhancer hypomethylation associates with oncogenic activation in early tumorigenesis. These results support a causal link between aberrant enhancer hypomethylation and C/EBPβ over-expression, thereby contributing to hepatocarcinogenesis through global transcriptional reprogramming.
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