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Correcting a patient-specific Rhodopsin mutation with adenine base editor in a mouse model.
Genome editing offers a great promise to treating human genetic diseases. To assess genome-editing-mediated therapeutic effects in vivo, an animal model is indispensable. The genomic disparities between mice and humans often impede the direct clinical application of genome-editing-mediated treatments using conventional mouse models. Thus, the generation of a mouse model with a humanized genomic segment containing a patient-specific mutation is highly sought after for translational research. In this study, we successfully developed a knockin mouse model for autosomal-dominant retinitis pigmentosa (adRP), designated as hT17M knockin, which incorporates a 75-nucleotide DNA segment with the T17M mutation (Rhodopsin-c.C50T; p.T17M). This model demonstrated significant reductions in electroretinogram amplitudes and exhibited disruptions in retinal structure. Subsequently, we administered an adeno-associated virus vectors carrying an adenine base editor (ABE) and a single-guide RNA specifically targeting the T17M mutation, achieving a peak correction rate of 39.7% at the RNA level and significantly improving retinal function in ABE-injected mice. These findings underscore that the hT17M knockin mouse model recapitulates the clinical features of adRP patients and exhibits therapeutic effects with ABE-mediated treatments. It offers a promising avenue for the development of gene-editing therapies for RP.
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Duchenne Muscular Dystrophy Gene Therapy.
Duchenne and Becker muscular dystrophies are allelic X-linked recessive neuromuscular diseases affecting both skeletal and cardiac muscles. Therefore, owing to their single X chromosome, the affected boys receive pathogenic gene mutations from their unknowing carrier mothers. Current pharmacological drugs are palliative that address the symptoms of the disease rather than the genetic cause imbedded in the Dystrophin gene DNA sequence. Therefore, alternative therapies like gene drugs that could address the genetic cause of the disease at its root are crucial, which include gene transfer/implantation, exon skipping, and gene editing. Presently, it is possible through genetic reprogramming to engineer AAV vectors to deliver certain therapeutic cargos specifically to muscle or other organs regardless of their serotype. Similarly, it is possible to direct the biogenesis of exosomes to carry gene editing constituents or certain therapeutic cargos to specific tissue or cell type like brain and muscle. While autologous exosomes are immunologically inert, it is possible to camouflage AAV capsids, and lipid nanoparticles to evade the immune system recognition. In this review, we highlight current opportunities for Duchenne muscular dystrophy gene therapy, which has been known thus far as an incurable genetic disease. This article is a part of Gene Therapy of Rare Genetic Diseases thematic issue.
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Development of a Haploid-Inducer Mediated Genome Editing System for Accelerating Maize Breeding.
Crop breeding aims to generate pure inbred lines with multiple desired traits. Doubled haploid (DH) and genome editing using CRISPR/Cas9 are two powerful game-changing technologies in crop breeding. However, both of them still fall short for rapid generation of pure elite lines with integrated favorable traits. Here, we report the development of a Haploid-Inducer Mediated Genome Editing (IMGE) approach, which utilizes a maize haploid inducer line carrying a CRISPR/Cas9 cassette targeting for a desired agronomic trait to pollinate an elite maize inbred line and to generate genome-edited haploids in the elite maize background. Homozygous pure DH lines with the desired trait improvement could be generated within two generations, thus bypassing the lengthy procedure of repeated crossing and backcrossing used in conventional breeding for integrating a desirable trait into elite commercial backgrounds.
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Targeting human oncogenic viruses with CRISPR/Cas: New therapeutic opportunities and challenges.
CRISPR/Cas systems, initially characterized as bacterial adaptive immune mechanisms, have rapidly emerged as precise and versatile genome-editing tools with significant potential for antiviral research and therapeutic development. This review highlights the role of CRISPR/Cas systems in targeting persistent and human oncogenic viruses, including HPV, HBV, HCV, EBV, KSHV, HTLV-1, and MCPyV, as well as HIV, which may indirectly contribute to cancer through immune dysregulation. Many of these viruses can integrate into the host genome or persist as chronic or latent infections, contributing to cancers for which curative options are limited. CRISPR-based strategies enable the excision of integrated proviral DNA, disruption of viral replication, targeted silencing of viral transcripts, and modulation of host tumor-suppressor pathways. Cas9 efficiently targets DNA viruses, such as HBV and HPV, whereas RNA-targeting Cas13 allows precise silencing of RNA viruses, like HCV. Editing T-cell receptors, including CCR5 and CXCR4, offers the potential for long-term resistance to HIV. CRISPR-based preclinical studies indicate the potential to disrupt HBV cccDNA, suppress EBV and KSHV latency gene expression, and inactivate HTLV-1 oncogenes, thereby potentially reducing viral persistence and oncogenic progression. Despite these advances, challenges remain regarding off-target effects, delivery efficiency, immune responses, and ethical considerations. Innovations such as high-fidelity Cas variants, base and prime editing, and non-viral delivery systems are expected to enhance both safety and therapeutic precision. This review provides an overview of viral life cycles, oncogenic pathways, and therapeutic vulnerabilities of human oncogenic viruses and CRISPR-based genome-editing approaches under investigation for viral elimination and cancer therapy.
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Improved selection of zebrafish CRISPR editing by early next-generation sequencing based genotyping.
Despite numerous prior attempts to improve knock-in (KI) efficiency, the introduction of precise base pair substitutions by the CRISPR-Cas9 technique in zebrafish remains challenging. In our efforts to generate KI zebrafish models of human CACNA1C mutations, we have tested the effect of several CRISPR determinants on KI efficiency across two sites in a single gene and developed a novel method for early selection to ameliorate KI efficiency. We identified optimal KI conditions for Cas9 protein and non-target asymmetric PAM-distal single stranded deoxynucleotide repair templates at both cacna1c sites. An effect of distance to the cut site on the KI efficiency was only observed for a single repair template conformation at one of the two sites. By combining minimally invasive early genotyping with the zebrafish embryo genotyper (ZEG) device and next-generation sequencing, we were able to obtain an almost 17-fold increase in somatic editing efficiency. The added benefit of the early selection procedure was particularly evident for alleles with lower somatic editing efficiencies. We further explored the potential of the ZEG selection procedure for the improvement of germline transmission by demonstrating germline transmission events in three groups of pre-selected embryos.
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Naturally Occurring Off-Switches for CRISPR-Cas9.
CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These "anti-CRISPRs" were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable "off-switches" for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. VIDEO ABSTRACT.
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Enhancement of CRISPR-Cas12a system through universal circular RNA design.
Precise control of Cas12a activity is crucial to address incompatibility in isothermal amplification-CRISPR-Cas12a one-pot nucleic acid detection. We developed a light-triggerable circular RNA system for dynamic LbCas12a regulation. By employing circular CRISPR guide RNA (crRNA) or a split circular universal direct repeat region with a replaceable spacer, we resolved the incompatibility between isothermal amplification and CRISPR detection. This system demonstrated robust performance in detecting trace nucleic acids in clinical samples. Furthermore, DNA modifications on circular crRNA enabled CRISPR-Cas12a regulation via base excision repair (BER) enzymes, offering potential for BER enzyme detection and modulation of LbCas12a cleavage activity by BER enzymes. This programmable strategy holds promise for selective gene editing in cells with elevated BER enzyme expression, such as uracil DNA glycosylase (UDG) in colon cancer cells. The circular RNA-assisted approach represents a resource-efficient method with significant potential for medical diagnostics and future clinical gene therapy applications.
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Engineered exosomes for CRISPR/Cas delivery to overcome oncogene-driven drug resistance.
Engineered exosomes are emerging as biocompatible nanocarriers for delivering CRISPR/Cas components to resistant tumor cells, enabling targeted disruption of oncogenic drivers and resistance-associated pathways. Engineered exosomes offer several delivery-platform advantages, including biocompatibility, membrane-mediated cargo protection, programmable tumor targeting, and potential tissue penetration. Selection of the CRISPR modality, Cas9 ribonucleoprotein, mRNA, base editor, or prime editor, depends on payload size, stability, editing duration, endosomal escape, and nuclear delivery requirements. Therapeutically, these systems may disrupt oncogenic drivers, inhibit resistance pathways, restore tumor-suppressor activity, and re-sensitize tumors to targeted therapy, chemotherapy, or immunotherapy. Clinical translation will require scalable manufacturing, reproducible cargo loading, standardized characterization, validated potency assays, off-target control, and clearly defined regulatory pathways. The goal of this review is to outline a realistic pathway featuring proof-of-concept research, through discoveries to the creation of manufacturable, safe, and effective exosome/CRISPR therapeutics that can trigger durable therapeutic responses in resistant malignancies.
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CRISPR/Cas genome editing in soybean: challenges and new insights to overcome existing bottlenecks.
BACKGROUND: Soybean is a worldwide-cultivated crop due to its applications in the food, feed, and biodiesel industries. Genome editing in soybean began with ZFN and TALEN technologies; however, CRISPR/Cas has emerged and shortly became the preferable approach for soybean genome manipulation since it is more precise, easy to handle, and cost-effective. Recent reports have focused on the conventional Cas9 nuclease, Cas9 nickase (nCas9) derived base editors, and Cas12a (formally Cpf1) as the most commonly used genome editors in soybean. Nonetheless, several challenges in the complex plant genetic engineering pipeline need to be overcome to effectively edit the genome of an elite soybean cultivar. These challenges include (1) optimizing CRISPR cassette design (i.e., gRNA and Cas promoters, gRNA design and testing, number of gRNAs, and binary vector), (2) improving transformation frequency, (3) increasing the editing efficiency ratio of targeted plant cells, and (4) improving soybean crop production. AIM OF REVIEW: This review provides an overview of soybean genome editing using CRISPR/Cas technology, discusses current challenges, and highlights theoretical (insights) and practical suggestions to overcome the existing bottlenecks. KEY SCIENTIFIC CONCEPTS OF REVIEW: The CRISPR/Cas system was discovered as part of the bacterial innate immune system. It has been used as a biotechnological tool for genome editing and efficiently applied in soybean to unveil gene function, improve agronomic traits such as yield and nutritional grain quality, and enhance biotic and abiotic stress tolerance. To date, the efficiency of gRNAs has been validated using protoplasts and hairy root assays, while stable plant transformation relies on Agrobacterium-mediated and particle bombardment methods. Nevertheless, most steps of the CRISPR/Cas workflow require optimizations to achieve a more effective genome editing in soybean plants.
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Targeting transthyretin by one Cas9 variant with superfidelity and broad compatibility.
Amyloid transthyretin (ATTR) amyloidosis is a fatal disease caused by the accumulation of misfolded transthyretin proteins. Although knocking down the TTR gene by CRISPR-Cas9 represents a promising strategy for treating ATTR amyloidosis, its efficiency and safety remain to be further investigated. Here, we report a systematic investigation of SpCas9-based TTR editing. Besides the target site, wild-type SpCas9 and the reported variants induced extensive off-target edits. To improve the fidelity, we performed structural analysis and designed a series of SpCas9 variants. Studies demonstrated that SpCas9-Mut5 is an ultrahigh-fidelity variant, which induces extremely low levels of off-target edits and translocations without substantial impairment of on-target editing activity. SpCas9-Mut5 is compatible with the adenine base editor (ABE) system, markedly reducing off-target edits and narrowing the editing window. In conclusion, our study suggests that SpCas9-Mut5 is an excellent candidate for TTR gene editing. Besides ATTR amyloidosis, SpCas9-Mut5 and its derivative ABE could be widely used in the treatment of other diseases.
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