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ABE9 fused to SpRY Cas9 nickase enables precise generation of bystander free mouse models.
Point mutations cause many genetic disorders, but modelling them in organisms is technically challenging. Creating mouse models that mimic these mutations is crucial for establishing a causal relationship between mutations and disease phenotype, thereby supporting the development of therapeutic strategies. Adenine base editors (ABEs) can correct single-nucleotide variants (SNVs) in disease modelling without double-stranded breaks (DSBs) or donor DNA, achieving higher product purity than traditional Cas9 methods. Earlier ABE techniques faced issues like limited targetability, bystander editing, and off-target effects. By combining two editor advancements, we introduced and tested ABE9-SpRY, an improved ABE variant fused with a PAM-flexible SpRY-Cas9 nickase. Our results show that ABE9-SpRY effectively generates three out of four targeted A-to-G mutations in mouse embryos, achieving desired editing efficiencies of up to 96% in individual adult founder mice. Furthermore, we observe fewer off-target events at predicted DNA sites in mouse embryos and in an orthogonal R-loop assay compared with ABE8e-SpRY. ABE9-SpRY also enhances product purity in mouse embryos under pooled sgRNA injections and, as a proof-of-concept, at a single endogenous locus in human induced pluripotent stem cells (hiPSCs), relative to ABE8e-SpRY. Our findings support ABE9-SpRY's precision at the loci tested and PAM-flexible versatility. Although performance remains sequence-dependent, these data support ABE9-SpRY as a PAM-flexible tool for generating precise point-mutation models where bystander editing is a concern.
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Generation of knock-in primary human T cells using Cas9 ribonucleoproteins.
T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently "knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4(+) T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.
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Precise A·T to G·C Base Editing in the Rice Genome.
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Advances in Gene Therapy for Sickle Cell Disease: From Preclinical Innovations to Clinical Implementation and Access Challenges.
Sickle cell disease (SCD) is a hereditary blood disorder caused by a specific mutation in the β-globin gene, leading to the production of hemoglobin S, which deforms red blood cells, causing occlusion in small blood vessels. This results in pain, anemia, organ damage, infections, and increased stroke risk. Treatment options, including disease-modifying therapies and curative hematopoietic stem cell transplants, have limited accessibility. Recently, autologous gene therapy has emerged as a promising curative option, particularly for SCD. Gene editing techniques such as CRISPR, base editing, and prime editing offer potential to correct this mutation. In this review, we discuss recent preclinical studies and clinical trials of gene and cell therapies, focusing on the progress of FDA-approved treatments like Lyfgenia and Casgevy. We also examine the many challenges, including accessibility, safety, and long-term efficacy, which continue to shape the future of SCD gene therapy.
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Protocol for arrayed gRNA screening by base editors in mammalian cell lines using lentiviral system.
Base editing, a CRISPR-based genome engineering technique, enables precise single-nucleotide modifications while minimizing double-strand breaks. Here, we present a protocol for arrayed mutagenesis using base editors to identify regulatory elements within the gamma-globin locus. We describe steps for guide RNA (gRNA) cloning into lentiviral vectors, establishing stable cell lines with base editor expression, transducing gRNAs, and assessing editing efficiency. This protocol can be applied to diverse genomic regions and cell lines for arrayed screening, facilitating genetic research, and target discovery. For complete details on the use and execution of this protocol, please refer to Ravi et al. (2022)1.
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Ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing as a therapy for cardiac disease.
CRISPR-Cas9 gene editing is emerging as a prospective therapy for genomic mutations. However, current editing approaches are directed primarily toward relatively small cohorts of patients with specific mutations. Here, we describe a cardioprotective strategy potentially applicable to a broad range of patients with heart disease. We used base editing to ablate the oxidative activation sites of CaMKIIδ, a primary driver of cardiac disease. We show in cardiomyocytes derived from human induced pluripotent stem cells that editing the CaMKIIδ gene to eliminate oxidation-sensitive methionine residues confers protection from ischemia/reperfusion (IR) injury. Moreover, CaMKIIδ editing in mice at the time of IR enables the heart to recover function from otherwise severe damage. CaMKIIδ gene editing may thus represent a permanent and advanced strategy for heart disease therapy.
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Retinal organoid and gene editing for basic and translational research.
The rapid evolution of two technologies has greatly transformed the basic, translational, and clinical research in the mammalian retina. One is the retinal organoid (RO) technology. Various induction methods have been created or adapted to generate species-specific, disease-specific, and experimental-targeted retinal organoids (ROs). The process of generating ROs can highly mimic the in vivo retinal development, and consequently, the ROs resemble the retina in many aspects including the molecular and cellular profiles. The other technology is the gene editing, represented by the classical CRISPR-Cas9 editing and its derivatives such as prime editing, homology independent targeted integration (HITI), base editing and others. The combination of ROs and gene editing has opened up countless possibilities in the study of retinal development, pathogenesis, and therapeutics. We review recent advances in the ROs, gene editing methodologies, delivery vectors, and related topics that are particularly relevant to retinal studies.
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Rare immune diseases paving the road for genome editing-based precision medicine.
Clustered regularly interspaced short palindromic repeats (CRISPR) genome editing platform heralds a new era of gene therapy. Innovative treatments for life-threatening monogenic diseases of the blood and immune system are transitioning from semi-random gene addition to precise modification of defective genes. As these therapies enter first-in-human clinical trials, their long-term safety and efficacy will inform the future generation of genome editing-based medicine. Here we discuss the significance of Inborn Errors of Immunity as disease prototypes for establishing and advancing precision medicine. We will review the feasibility of clustered regularly interspaced short palindromic repeats-based genome editing platforms to modify the DNA sequence of primary cells and describe two emerging genome editing approaches to treat RAG2 deficiency, a primary immunodeficiency, and FOXP3 deficiency, a primary immune regulatory disorder.
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CRISPR-Combo-mediated orthogonal genome editing and transcriptional activation for plant breeding.
CRISPR-Cas nuclease systems, base editors, and CRISPR activation have greatly advanced plant genome engineering. However, the combinatorial approaches for multiplexed orthogonal genome editing and transcriptional regulation were previously unexploited in plants. We have recently established a single Cas9 protein-based CRISPR-Combo platform, enabling efficient multiplexed orthogonal genome editing (double-strand break-mediated genome editing or base editing) and transcriptional activation in plants via engineering the single guide RNA (sgRNA) structure. Here, we provide step-by-step instructions for constructing CRISPR-Combo systems for speed breeding of transgene-free, genome-edited Arabidopsis plants and enhancing rice regeneration with more heritable targeted mutations in a hormone-free manner. We also provide guidance on designing efficient sgRNA, Agrobacterium-mediated transformation of Arabidopsis and rice, rice regeneration without exogenous plant hormones, gene editing evaluation and visual identification of transgene-free Arabidopsis plants with high editing activity. With the use of this protocol, it takes ~2 weeks to establish the CRISPR-Combo systems, 4 months to obtain transgene-free genome-edited Arabidopsis plants and 4 months to obtain rice plants with enrichment of heritable targeted mutations by hormone-free tissue culture.
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CRISPR-Cas9: Genome Engineering and Future Vaccine Applications.
The CRISPR-Cas9 system, a transformative genome engineering tool derived from prokaryotic adaptive immunity, is reshaping the landscape of biological research and therapeutic development. This review provides a critical synthesis of its rapidly evolving, yet underexplored, application in rational vaccine design. We analyze how CRISPR-Cas9 and its derivative platforms (including base editing, prime editing, and CRISPRi/a) are being repurposed from therapeutic gene editing to become indispensable assets in vaccinology. This transition is powered by the convergence of CRISPR-mediated precision with synthetic biology, enabling the rapid engineering of novel vaccine vectors and attenuated strains, the precise optimization of antigen sequences for enhanced breadth and potency, and the direct modulation of host immune responses. Notwithstanding this potential, significant technical and translational hurdles persist, including off-target editing risks, delivery inefficiencies in vivo, and unresolved regulatory pathways for genetically modified vaccines. We detail these mechanisms and evaluate the current preclinical and clinical landscape, while addressing persistent challenges in safety, delivery, and scalability. By delineating these advances and obstacles, this review outlines a forward-looking framework for leveraging CRISPR technology to create programmable, precision vaccines against emerging and re-emerging pathogens, moving the field beyond empirical methods toward a new paradigm of rational immunization.
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