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Optimization of genome editing strategies for targeted integration at chicken sex chromosome loci.
The precise targeted integration of large exogenous genes into chicken sex chromosomes is of particular interest for sex-linked trait manipulation, sex-control breeding, and the development of avian bioreactor models. However, efficient targeted integration at sex chromosome loci remains technically challenging, and optimized editing strategies for these loci are still lacking. To improve targeted genome editing at two previously identified chicken sex chromosome safe-harbor loci, EE0.6 and NC_006127.4, this study systematically evaluated and optimized key parameter affecting editing efficiency. First, we evaluated the effects of different sgRNA combinations on targeted knockout efficiency, establishing the advantage of a dual-sgRNA/Cas9 architecture, which achieved knockout efficiencies of 86.67% and 75.00% at the EE0.6 and NC_006127.4 loci, respectively. We next introduced the Cas9 nickase (Cas9n) system, which has previously been reported to exhibit improved editing specificity, and evaluated its performance at both target loci. Quantitative analysis showed that the dual-sgRNA/Cas9 targeting system successfully mediated the precise targeted integration of a 1.1-kb SV40-mCherry reporter cassette, reaching 100% (28/28) at the EE0.6 locus and 80.00% (20/25) at the NC_006127.4 locus. Based on this result, this study further investigated the effects of donor homology arm (HA) lengths (200 bp, 600 bp, and, 1000 bp) and vector topologies (circular and linearized) on targeted knock-in efficiency. The results revealed that in the circular donor system, the optimal HA lengths for the EE0.6 and NC_006127.4 loci were 200 bp (50.4% ± 4.4%) and 600 bp (30.1% ± 1.2%), respectively. However, upon the introduction of linearized donors with free ends, the knock-in efficiency of the exogenous target fragment was significantly enhanced, and its HA length preference underwent a significant reversal. The optimal HA length for EE0.6 was extended to 600 bp (78.9% ± 1.0%), whereas that for NC_006127.4 was shortened to 200 bp (49.9% ± 0.4%). In summary, this study established an efficient targeted integration strategy for chicken sex chromosome loci. The optimized system provides a foundation for future applications in sex-linked breeding and avian bioreactor development.
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Genome Editing in Ferret Airway Epithelia Mediated by CRISPR/Nucleases Delivered with Amphiphilic Shuttle Peptides.
Gene editing strategies are attractive for treating genetic pulmonary diseases such as cystic fibrosis (CF). However, challenges have included the development of safe and effective vector systems for gene editing of airway epithelia and model systems to report their efficiency and durability. The domestic ferret (Mustela putorius furo) has a high degree of conservation in lung cellular anatomy with humans, and has served as an excellent model for many types of lung diseases, including CF. In this study, we evaluated the efficiency of amphiphilic shuttle peptide S10 for protein delivery and gene editing using SpCas9, and AsCas12a (Cpf1) ribonucleoproteins (RNPs). These approaches were evaluated in proliferating ferret airway basal cells, polarized airway epithelia in vitro, and lungs in vivo, by accessing the editing efficiency using reporter ferrets and measuring indels at the ferret CFTR locus. Our results demonstrate that shuttle peptides efficiently enable delivery of reporter proteins/peptides and gene editing SpCas9 or Cpf1 RNP complexes to ferret airway epithelial cells in vitro and in vivo. We measured S10 delivery efficiency of green fluorescent protein (GFP)-nuclear localization signal (NLS) protein or SpCas9 RNP into ferret airway basal cells and fully differentiated ciliated and nonciliated epithelial cells in vitro. In vitro and in vivo gene editing efficiencies were determined by Cas/LoxP-gRNA RNP-mediated conversion of a ROSA-TG Cre recombinase reporter using transgenic primary cells and ferrets. S10/Cas9 RNP was more effective, relative to S10/Cpf1 RNP at gene editing of the ROSA-TG locus. Intratracheal lung delivery of the S10 shuttle combined with GFP-NLS protein or D-Retro-Inverso (DRI)-NLS peptide demonstrated efficiencies of protein delivery that were ∼3-fold or 14-fold greater, respectively, than the efficiency of gene editing at the ROSA-TG locus using S10/Cas9/LoxP-gRNA. Cpf1 RNPs was less effective than SpCas9 at gene editing of LoxP locus. These data demonstrate the feasibility of shuttle peptide delivery of Cas RNPs to the ferret airways and the potential utility for developing ex vivo stem cell-based and in vivo gene editing therapies for genetic pulmonary diseases such as CF.
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Topology-Engineered Guide RNAs for Programmable Control of CRISPR/Cas Activity.
CRISPR/Cas systems have transformed genome editing, yet achieving precise temporal and conditional control remains challenging. Traditional strategies involving linear guide RNAs (gRNAs) modified with multiple chemical groups throughout their strands often face limitations such as heterogeneous reaction outcomes, irreversibility, and variable editing efficiencies. To overcome these issues, topology-engineered guide RNAs (TE-gRNAs) have emerged, featuring defined structural architectures including polymeric, circular, and dendrimer-like topologies that enable precise spatial control, reversibility, and programmable activation of CRISPR activity. By selectively incorporating physical or chemically responsive linkers and stimuli-sensitive groups at specific sites, TE-gRNAs facilitate dynamic and conditional genome editing that can be activated or deactivated with external triggers such as light or chemical signals. These engineered RNA structures significantly improve synthesis feasibility, stability, reduce off-target effects, and provide unprecedented control over gene editing processes. Recent advancements in TE-gRNAs demonstrate their broad applicability in synthetic biology, functional genomics, and therapeutic interventions, highlighting their potential to achieve precise spatiotemporal modulation of CRISPR systems. This review summarizes the current strategies, benefits, and challenges associated with TE-gRNAs, and discusses future directions for enhancing their performance and utility in complex genome editing applications.
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Therapeutic applications of gene editing in chronic liver diseases: an update.
Innovative genome editing techniques developed in recent decades have revolutionized the biomedical research field. Liver is the most favored target organ for genome editing owing to its ability to regenerate. The regenerative capacity of the liver enables ex vivo gene editing in which the mutated gene in hepatocytes isolated from the animal model of genetic disease is repaired. The edited hepatocytes are injected back into the animal to mitigate the disease. Furthermore, the liver is considered as the easiest target organ for gene editing as it absorbs almost all foreign molecules. The mRNA vaccines, which have been developed to manage the COVID-19 pandemic, have provided a novel gene editing strategy using Cas mRNA. A single injection of gene editing components with Cas mRNA is reported to be efficient in the treatment of patients with genetic liver diseases. In this review, we first discuss previously reported gene editing tools and cases managed using them, as well as liver diseases caused by genetic mutations. Next, we summarize the recent successes of ex vivo and in vivo gene editing approaches in ameliorating liver diseases in animals and humans. [BMB Reports 2022; 55(6): 251-258].
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Development of a marker-free mutagenesis system using CRISPR-Cas9 in the pathogenic mould Aspergillus fumigatus.
Aspergillus fumigatus is a saprophytic fungal pathogen that is the cause of more than 300,000 life-threatening infections annually. Our understanding of pathogenesis and factors contributing to disease progression are limited. Development of rapid and versatile gene editing methodologies for A. fumigatus is essential. CRISPR-Cas9 mediated transformation has been widely used as a novel genome editing tool and has been used for a variety of editing techniques, such as protein tagging, gene deletions and site-directed mutagenesis in A. fumigatus. However, successful genome editing relies on time consuming, multi-step cloning procedures paired with the use of selection markers, which can result in a metabolic burden for the host and/or unintended transcriptional modifications at the site of integration. We have used an in vitro CRISPR-Cas9 assembly methodology to perform selection-free genome editing, including epitope tagging of proteins and site-directed mutagenesis. The repair template used during this transformation use 50 bp micro-homology arms and can be generated with a single PCR reaction or by purchasing synthesised single stranded oligonucleotides, decreasing the time required for complex construct synthesis.
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CRISPR/Cas9 Inhibits Multiple Steps of HIV-1 Infection.
CRISPR/Cas9 is an adaptive immune system where bacteria and archaea have evolved to resist the invading viruses and plasmid DNA by creating site-specific double-strand breaks in DNA. This study tested this gene editing system in inhibiting human immunodeficiency virus type 1 (HIV-1) infection by targeting the viral long terminal repeat and the gene coding sequences. Strong inhibition of HIV-1 infection by Cas9/gRNA was observed, which resulted not only from insertions and deletions (indels) that were introduced into viral DNA due to Cas9 cleavage, but also from the marked decrease in the levels of the late viral DNA products and the integrated viral DNA. This latter defect might have reflected the degradation of viral DNA that has not been immediately repaired after Cas9 cleavage. It was further observed that Cas9, when solely located in the cytoplasm, inhibits HIV-1 as strongly as the nuclear Cas9, except that the cytoplasmic Cas9 does not act on the integrated HIV-1 DNA and thus cannot be used to excise the latent provirus. Together, the results suggest that Cas9/gRNA is able to target and edit HIV-1 DNA both in the cytoplasm and in the nucleus. The inhibitory effect of Cas9 on HIV-1 is attributed to both the indels in viral DNA and the reduction in the levels of viral DNA.
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CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia.
Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.
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Epigenome Engineering Using dCas Systems for Biomedical Applications and Biotechnology: Current Achievements, Opportunities and Challenges.
Epigenome engineering, particularly utilizing CRISPR/dCas-based systems, is a powerful strategy to modulate gene expression and genome functioning without altering the DNA sequence. In this review we summarized current achievements and prospects in dCas-mediated epigenome editing, primarily focusing on its applications in biomedicine, but also providing a wider context for its applications in biotechnology. The diversity of CRISPR/dCas architectures is outlined, recent innovations in the design of epigenetic editors and delivery methods are highlighted, and the therapeutic potential across a wide range of diseases, including hereditary, neurodegenerative, and metabolic disorders, is examined. Opportunities for the application of dCas-based tools in animal, agricultural, and industrial biotechnology are also discussed. Despite substantial progress, challenges, such as delivery efficiency, specificity, stability of induced epigenetic modifications, and clinical translation, are emphasized. Future directions aimed at enhancing the efficacy, safety, and practical applicability of epigenome engineering technologies are proposed.
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The Next Generation of Molecular and Cellular Therapeutics for Inherited Retinal Disease.
Inherited retinal degenerations (IRDs) are a diverse group of conditions that are often characterized by the loss of photoreceptors and blindness. Recent innovations in molecular biology and genomics have allowed us to identify the causative defects behind these dystrophies and to design therapeutics that target specific mechanisms of retinal disease. Recently, the FDA approved the first in vivo gene therapy for one of these hereditary blinding conditions. Current clinical trials are exploring new therapies that could provide treatment for a growing number of retinal dystrophies. While the field has had early success with gene augmentation strategies for treating retinal disease based on loss-of-function mutations, many novel approaches hold the promise of offering therapies that span the full spectrum of causative mutations and mechanisms. Here, we provide a comprehensive review of the approaches currently in development including a discussion of retinal neuroprotection, gene therapies (gene augmentation, gene editing, RNA modification, optogenetics), and regenerative stem or precursor cell-based therapies. Our review focuses on technologies that are being developed for clinical translation or are in active clinical trials and discusses the advantages and limitations for each approach.
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A Roadmap toward Large-Scale Genome Editing in Crops.
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