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Found: 26 147
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Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing.
Cytosine base editors and adenine base editors (ABEs) can correct point mutations predictably and independent of Cas9-induced double-stranded DNA breaks (which causes substantial indel formation) and homology-directed repair (which typically leads to low editing efficiency). Here, we show, in adult mice, that a subretinal injection of a lentivirus expressing an ABE and a single-guide RNA targeting a de novo nonsense mutation in the Rpe65 gene corrects the pathogenic mutation with up to 29% efficiency and with minimal formation of indel and off-target mutations, despite the absence of the canonical NGG sequence as a protospacer-adjacent motif. The ABE-treated mice displayed restored RPE65 expression and retinoid isomerase activity, and near-normal levels of retinal and visual functions. Our findings motivate the further testing of ABEs for the treatment of inherited retinal diseases and for the correction of pathological mutations with non-canonical protospacer-adjacent motifs.
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Beyond prediction: The evolving role of artificial intelligence in genome editing.
Since the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system, genome-editing technologies have advanced rapidly, and the field has expanded substantially. In parallel, artificial intelligence (AI) has progressed at an unprecedented pace and is transforming genetic engineering. Early AI applications primarily addressed predictive tasks, including guide RNA (gRNA) activity, off-target effects, and editing outcomes for CRISPR-Cas nucleases, base editors, and prime editors. Recently, transformer-based protein language models (PLMs) and generative AI have expanded the role of AI beyond prediction to direct molecular design. Thus, AI-driven frameworks can now evolve genome-editing proteins, design molecular architectures, and generate de novo genome-editing enzymes. This review discusses how AI is reshaping genome engineering, from gRNA activity prediction to the design of next-generation editors.
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Germline Editing of Drosophila Using CRISPR-Cas9-Based Cytosine and Adenine Base Editors.
Target-AID, BE3, and ABE7.10 base editors fused to the catalytically modified Cas9 and xCas9(3.7) were tested for germline editing of the fruit fly Drosophila melanogaster. We developed a guide RNA-expressing construct, white-4gRNA, targeting splice sites in the white gene, an X-chromosome located gene. Using white-4gRNA flies and transgenic lines expressing Target-AID, BE3, and ABE7.10 base editors, we tested the efficiency of stable germline gene editing at three different temperatures. Classical Cas9 generating insertions/deletions by non-homologous end joining served as a reference. Our data indicate that gene editing is most efficient at 28°C, the highest temperature suitable for fruit flies. Finally, we created a new allele of the core circadian clock gene timeless using Target-AID. This base edited mutant allele timSS308-9FL had a disrupted circadian clock with a period of ∼29 h. The white-4gRNA expressing fly can be used to test new generations of base editors for future applications in Drosophila.
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An Expert Overview on Therapies in Non-Transfusion-Dependent Thalassemia: Classical to Cutting Edge in Treatment.
The thalassemia issue is a growing worldwide health concern that anticipates the number of patients suffering from the disease will soon increase significantly. Patients with β-thalassemia intermedia (β-TI) manifest mild to intermediate levels of anemia, which is a reason for it to be clinically located between thalassemia minor and β-thalassemia major (β-TM). Notably, the determination of the actual rate of β-TI is more complicated than β-TM. The leading cause of this illness could be partial repression of β-globin protein production; accordingly, the rate of β-globin gene repression is different in patients, and the gene repression intensity creates a different clinical status. This review article provides an overview of functional mechanisms, advantages, and disadvantages of the classic to latest new treatments for this group of patients, depending on the disease severity divided into the typical management strategies for patients with β-TI such as fetal hemoglobin (Hb) induction, splenectomy, bone marrow transplantation (BMT), transfusion therapy, and herbal and chemical iron chelators. Recently, novel erythropoiesis-stimulating agents have been added. Novel strategies are subclassified into molecular and cellular interventions. Genome editing is one of the efficient molecular therapies for improving hemoglobinopathies, especially β-TI. It encompasses high-fidelity DNA repair (HDR), base and prime editing, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 procedure, nuclease-free strategies, and epigenetic modulation. In cellular interventions, we mentioned the approach pattern to improve erythropoiesis impairments in translational models and patients with β-TI that involve activin II receptor traps, Janus-associated kinase 2 (JAK2) inhibitors, and iron metabolism regulation.
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High-Throughput Profiling of Cas12a Orthologues and Engineered Variants for Enhanced Genome Editing Activity.
CRISPR/Cas12a (formerly Cpf1), an RNA-guided endonuclease of the Class II Type V-A CRISPR system, provides a promising tool for genome engineering. Over 10 Cas12a orthologues have been identified and employed for gene editing in human cells. However, the functional diversity among emerging Cas12a orthologues remains poorly explored. Here, we report a high-throughput comparative profiling of editing activities across 16 Cas12a orthologues in human cells by constructing genome-integrated, self-cleaving, paired crRNA-target libraries containing >40,000 guide RNAs. Three Cas12a candidates exhibited promising potential owing to their compact structures and editing efficiency comparable with those of AsCas12a and LbCas12a, which are well characterized. We generated three arginine substitution variants (3Rv) via structure-guided protein engineering: BsCas12a-3Rv (K155R/N512R/K518R), PrCas12a-3Rv (E162R/N519R/K525R), and Mb3Cas12a-3Rv (D180R/N581R/K587R). All three Cas12a variants showed enhanced editing activities and expanded targeting ranges (NTTV, NTCV, and TRTV) compared with the wild-type Cas12a effectors. The base preference analysis among the three Cas12a variants revealed that PrCas12a-3Rv shows the highest activity at target sites with canonical PAM TTTV and non-canonical PAM TTCV, while Mb3Cas12a-3Rv exhibits recognition features distinct from the others by accommodating for more nucleotide A at position -3 for PAM TATV and at position -4 for PAM ATCV. Thus, the expanded Cas12a toolbox and an improved understanding of Cas12a activities should facilitate their use in genome engineering.
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CRISPR/cas genome editing for neurodegenerative diseases: Mechanisms, therapeutic advances, and clinical prospects.
Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS), Spinocerebral Ataxia (SCA), and Huntington's disease (HD) are major global health challenges. Current treatments are only symptomatic and do not address the underlying pathogenic genetic mechanisms. The development of the CRISPR/Cas genome editing technologies, has increased possibilities for targeted repair of pathological mutations. CRISPR/Cas9, Cas12, and Cas13 systems enable targeted editing and transcriptome modulation in various preclinical models. CRISPR/Cas9 disruption of mutant APP, Tau, and LRRK2 genes, reducing toxic protein aggregration in AD models has restored normal genetic function. While correction of CAG nucleotide repeats in HD, and reduction of alpha-synuclein expression in PD. RNA targeting systems like Cas13 offers additional therapeutics potential by selectively degrading disease assciated transcript without altering genomic DNA. Advancements in engineered Cas variants with enhanced specificity, such as SpCas9-HF1, base editors and prime editors, with innovative delivery strategies including adeno-associated virus (AAVs) and nanoparticle-based systems, have improved genome editing. However, challenges remain, including off-target effects, mosaicism, and delivery across the BBB, and long-term safety. Ethical consideration focuses on somatic versus germline editing, equitable access, and regulatory oversight. While somatic editing shows acceptance in treating neurological disorders. Germline interventions face strict regulations due to potential multigeneration impacts. Collectively, these technologies are the vanguard of precision molecular medicine, advancing from symptom management towards potentially curative gene therapies for neurological disorders.
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CRISPR/Cas9-Mediated Genome Editing of Trichoderma reesei.
In this protocol, we describe the establishment of a CRISPR/Cas9 system in Trichoderma reesei by generating a specific, codon-optimized Cas9-expressing strain and by in vitro transcription of a gRNA. This system induces mutagenesis or introduces a gene in a targeted way based on PEG-mediated protoplast transformation. Up to three targets, multiplexed genome editing can be obtained in one transformation.
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'Base editors' open new way to fix mutations.
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CRISPR/Cas9 genome editing-based preclinical theranostics, biomarkers and drug delivery systems for cancer applications.
Cancer development is driven by diverse genetic aberrations, underscoring the importance of innovative approaches like gene therapy for effective treatment. The CRISPR/Cas9 gene-editing system has all the makings of a game-changing technique for future disease treatment, owing to its pinpoint accuracy and efficiency in deleting disease-causing genes or correcting damaging base mutations. A number of efficient Cas9 variants and derivatives were recently designed to tackle the intricate genomic modifications that accompany illnesses. In addition, CRISPR/Cas9 based systems are increasingly explored for biomarker sensing and cancer diagnostics. Early identification, real-time monitoring, and therapy stratification are made possible by CRISPR-driven biosensors, which can detect circulating tumor DNA, microRNAs, or exosomal RNA with high specificity and sensitivity. Furthermore, a variety of stimuli-responsive delivery strategies, including chemical and peptide-assisted systems, light-activated mechanisms, glutathione-sensitive carriers, and pH-responsive platforms, have been explored to improve intracellular release efficiency thereby enhancing the precision of CRISPR/Cas9-mediated gene editing in cancer therapy. The CRISPR/Cas9-enabled theranostic platforms employ engineered nanocarriers to simultaneously deliver gene-editing tools and imaging agents, thereby facilitating synchronized treatment monitoring and improved therapeutic precision. This review emphasizes the transformative potential of CRISPR/Cas9-integrated theranostics, which combine targeted gene editing with advanced imaging for enhanced therapeutic monitoring and efficacy in cancer treatment.
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Gene editing of the GJB2 locus in porcine embryos using CRISPR/Cas9 and cytosine base editors: toward a model of congenital deafness.
Mutations in the GJB2 gene, which encodes Connexin 26 (Cx26), are responsible for the majority of cases of non-syndromic congenital hearing loss in humans. While murine GJB2 knockout models have provided mechanistic insight, anatomical and physiological differences limit their translational relevance. Pigs represent a valuable large-animal model because their auditory anatomy and maturation closely resemble those of humans. This study compared two genome-editing approaches to disrupt GJB2 in porcine oocytes before fertilization: (1) electroporation with CRISPR/Cas9 ribonucleoprotein and (2) microinjection with cytosine base editor (BE3) and single-guide RNAs (sgRNAs). Electroporation produced high mutation rates (70–90%) across three concentrations of Cas9/sgRNA but yielded mostly heterozygous or mosaic blastocysts, with limited homozygous knockouts (< 4%). BE3 achieved precise cytosine-to-thymine conversions that introduced premature stop codons, reaching up to 47% total editing and 20% homozygous nonsense alleles. However, blastocyst formation declined at higher component concentrations. Overall, BE3 produced more predictable mutations than conventional CRISPR/Cas9, although embryo developmental competence was dose-dependent. Both methods effectively targeted GJB2 and demonstrated feasibility of pre-fertilization genome editing in porcine oocytes. These findings establish the groundwork for generating GJB2-deficient pigs as translational models of Cx26-related congenital deafness and for future evaluation of gene-therapy strategies in a large-animal system.
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