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Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish.
While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA protospacer adjacent motif, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic an mutation of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes including cbl. With this last target, we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease-associated mutations.
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Elucidating the genetic mechanisms governing cytosine base editing outcomes through CRISPRi screens.
Cytosine base editors enable programmable and efficient genome editing using an intermediate featuring a U•G mismatch across from a DNA nick. This intermediate facilitates two major outcomes, C•G to T•A and C•G to G•C point mutations, and it is not currently well-understood which DNA repair factors are involved. Here, we couple reporters for cytosine base editing activity with knockdown of 2015 DNA processing genes to identify genes involved in these two outcomes. Our data suggest that mismatch repair factors facilitate C•G to T•A outcomes, while C•G to G•C outcomes are mediated by RFWD3, an E3 ubiquitin ligase. We also propose that XPF, a 3'-flap endonuclease, and LIG3, a DNA ligase, are involved in repairing the intermediate back to the original C•G base pair. Our results demonstrate that competition and collaboration among different DNA repair pathways shape cytosine base editing outcomes.
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Gene therapy for sickle cell disease and thalassemia.
PURPOSE OF REVIEW: Thalassemia and sickle cell disease are among the most frequent monogenic hereditary diseases. Access to transfusions, iron chelation therapies and drugs such as hydroxyurea have improved life expectancy and quality of life. However, these diseases still cause significant disability. The first available curative therapy, bone marrow transplantation, is unfortunately not feasible for all patients. Over the past decade, numerous studies have focused on finding new curative therapies, and many clinical trials have evaluated different gene therapy approaches. RECENT FINDINGS: The therapeutic targets focus on adding functional copies of the gene encoding β-globin in defective CD34 + cells, mainly using lentiviral vectors directed towards HSCs. More recently, the focus has shifted to inducing fetal hemoglobin production at therapeutic levels or repairing the underlying molecular defect, using novel gene editing techniques involving CRISPR-Cas9, transcription activation-like effector protein nucleases, zinc finger nucleases and base editing. Preclinical and clinical studies now focus on optimizing how gene therapy is performed and delivered to reduce or eliminate myeloablative treatment and its potential adverse events. SUMMARY: In this review, we explore the potential to induce fetal hemoglobin production at therapeutic levels or to repair the underlying molecular defect that causes the disease genetically. Here, we review recent gene editing studies that are opening a new era in curative treatment for hemoglobinopathies.
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ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana.
Adenine base editor containing TadA8e (ABE8e) has been reported in rice. However, the application of ABE8e in other plant species has not been described, and the comparison between ABE8e and ABE7.10, which is widely used in plants, has also been poorly studied. Here, we developed the ABE8e with the polycistronic tRNA-gRNA expression cassette (PTG-ABE8e) and PTG-ABE7.10 and compared their A-to-G editing efficiencies using both transient and stable transformation in the allotetraploid Nicotiana benthamiana. We found that the editing efficiency of PTG-ABE8e was significantly higher than that of PTG-ABE7.10, indicating that ABE8e was more efficient for A-to-G conversion in N. benthamiana. We further optimized the ABE8e editing efficiency by changing the sgRNA expression cassette and demonstrated that both PTG and single transcript unit (STU) enhanced ABE8e efficiency for A-to-G conversion in N. benthamiana. We also estimated the potential off-target effect of PTG-ABE8e at potential off-targeting sites predicted using an online tool in transgenic plants, and no off-target editing event was found for potential off-targeting sites selected, indicating that ABE8e could specifically facilitate A-to-G conversion. Our results showed that ABE8e with PTG structure was more suitable for A-to-G conversion in N. benthamiana and provided valuable clues for optimizing ABE tools in other plants.
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Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing.
It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.
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CRISPR technology in ovarian cancer research: advances from gene editing to precision diagnosis and therapy.
Ovarian cancer (OC) remains a leading cause of gynecological cancer mortality, driven by pronounced molecular heterogeneity, late diagnosis, and frequent acquisition of therapeutic resistance. The advent of CRISPR-Cas9 genome-editing technology has fundamentally transformed OC research by enabling precise, scalable, and unbiased interrogation of genetic drivers underlying tumor initiation, metastatic progression, drug resistance, and immune evasion. This review systematically summarizes the evolutionary trajectory of CRISPR-Cas9 platforms, from the discovery of the prokaryotic adaptive immune system to the development of high-fidelity nucleases, base editors, and prime editing systems. We then critically discuss the diversity of CRISPR-based library screening strategies, including CRISPR knockout (CRISPRko), CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), and base editing libraries, highlighting their respective advantages, limitations, and technical considerations for high-throughput functional genomics. A major focus is placed on the broad applications of these tools in OC research, such as the systematic discovery of novel oncogenic drivers and metastatic mediators, the functional dissection of chemotherapy and PARP inhibitor resistance mechanisms, the construction of genetically defined patient-derived organoid and in vivo models, and the development of CRISPR-based diagnostic platforms for early detection and liquid biopsy monitoring. Furthermore, we provide an in-depth overview of emerging delivery systems for OC, including engineered viral vectors, non‑viral nanoparticles, physical methods, and exosome‑based platforms, with an emphasis on their adaptation to the unique peritoneal microenvironment. Finally, we highlight key translational challenges that currently constrain clinical implementation, such as off‑target editing risks, tumor heterogeneity, immune barriers, and ethical considerations, and propose future directions to overcome these hurdles. Collectively, the integration of CRISPR‑based functional screening with multi‑omics profiling, single‑cell technologies, and precision medicine frameworks offers a powerful paradigm for advancing personalized therapeutic strategies and improving long‑term outcomes for patients with ovarian cancer.
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Boosting genome editing of non-coding sequences in plants with glycosylase-mediated multi-nucleotide deletion editors.
Compared with protein-coding sequences, non-coding sequences-such as promoters, untranslated regions (UTRs), microRNAs, and other regulatory ncRNAs-constitute the vast majority of the plant genome. They serve as critical regulators of gene expression and therefore represent promising targets for crop improvement. However, their study and manipulation remain challenging due to the lack of efficient tools for generating large genomic deletions. Here, we report that CRISPR-Cas9-based glycosylase base editors (gBEs) function primarily as highly efficient multi-nucleotide deletion editors (gMDEs) in plants, a role distinct from their predominant base-editing activity in mammals. This functional shift is likely driven by a preferential AP lyase repair pathway for glycosylase-generated abasic sites (apurinic/apyrimidinic or AP sites) in plant cells. Unlike its parental system, CRISPR-Cas9, gMDEs efficiently generate 6-20 bp deletions across protospacers in both rice and soybean. We demonstrate their versatility by generating a continuum of plant height variation through promoter editing of OsD18 and boosting grain size by disrupting regulatory elements in both the 5' and 3' UTRs of OsGLW7, which function through distinct regulatory mechanisms. This work establishes gMDEs as a versatile and precise genome editing platform for inducing multi-nucleotide deletions in plants, making them efficient tools for genetic perturbation, especially of non-coding sequences.
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SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition.
BACKGROUND: Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome. RESULTS: In this study, we thoroughly investigate the nuclease activity and the PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice. Our study shows that SpG nuclease favors NGD PAMs, albeit less efficiently than the previously described SpCas9-NG, and that SpRY nuclease achieves efficient editing across a wide range of genomic loci, exhibiting a preference of NGD as well as NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*Δ and adenosine deaminase TadA8e are generated, respectively. These constructs efficiently induce C-to-T and A-to-G conversions in the target genes toward various non-canonical PAMs, including non-G PAMs. Remarkably, high-frequency self-editing events (indels and DNA fragments deletion) in the integrated T-DNA fragments as a result of the nuclease activity of SpRY are observed, whereas the self-editing of SpRY nickase-mediated base editor is quite low in transgenic rice lines. CONCLUSIONS: The broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.
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Genetic Correction of the Most Common Mutation Causing Primary Hyperoxaluria Restores Enzyme Localization and Oxalate Metabolism.
Our research aimed to model primary hyperoxaluria type 1 in vitro using a stem cell model and assess the potential of adenine base editors in correcting the most common pathogenic AGXT genetic variant, c.508G>A (Gly170Arg), which leads to oxalate accumulation due to alanine-glyoxylate aminotransferase mislocalization. Patient-derived fibroblasts were induced to pluripotent stem cells, genetically corrected with adenine base editing, and subsequently differentiated into hepatocyte-like cells in parallel with their non-corrected isogenic counterparts. Enzyme localization was assessed through immunocytochemistry and confocal microscopy. The key metabolites associated with the disease were analyzed using liquid chromatography-mass spectrometry to evaluate the metabolic phenotype. Finally, lipid nanoparticle formulations were designed and tested as an in vivo-applicable delivery method for base editors. All induced pluripotent stem cell lines successfully differentiated into hepatocyte-like cells and expressed essential hepatocyte markers, including ALB, HNF1A, and AGXT. Adenine base editor-mediated genetic correction of the pathogenic AGXT mutation restored enzyme localization into peroxisomes and diminished oxalate accumulation without significant off-target effects. Base editor mRNA and AGXT variant targeting single guide RNA encapsulated within lipid nanoparticles mediated gene correction in the hepatocyte-like cell model. Using an in vitro model of primary hyperoxaluria type 1, we showed that base editor-mediated genetic correction of the most common hyperoxaluria-causing variant corrects enzyme mislocalization from mitochondria to peroxisomes and improves metabolic function. These results propose gene correction as a potential therapeutic approach to hyperoxaluria.
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CRISPR/Cas9-mediated one step bi-allelic change of genomic DNA in iPSCs and human RPE cells in vitro with dual antibiotic selection.
CRISPR/Cas9 causes double-stranded DNA breaks that can undergo DNA repair either via non-homologous end joining (NHEJ) or, in the presence of a template, homology-directed repair (HDR). HDR is typically used to insert a specific genetic modification into the genome but has low efficiency compared to NHEJ, which is lowered even further when trying to create a homozygous change. In this study we devised a novel approach for homozygous single base editing based on utilising simultaneously two donor DNA templates cloned in plasmids with different antibiotic resistant genes. The donor templates were co-transfected alongside the CRISPR/Cas9 machinery into cells and a double antibiotic selection was optimised and allowed the isolation of viable desired clones. We applied the method for obtaining isogenic cells homozygous for variant B cystatin C, a recessive risk factor for age-related macular degeneration and Alzheimer's disease, in both induced Pluripotent Stem Cells (iPSCs) and a human RPE cell line. Bi-allelic gene edited clones were validated by sequencing, demonstrating that the double antibiotic templates approach worked efficiently for both iPSCs and human differentiated cells. We propose that this one step gene editing approach can be used to improve the specificity and frequency of introducing homozygous modifications in mammalian cells.
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