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Advances in mitochondrial DNA base editing technology.
Mitochondria, the energy factories of higher eukaryotes, provide energy (ATP) for life activities through aerobic respiration. They possess their own genome, mitochondrial DNA (mtDNA), which encodes 37 genes. Mutations in mtDNA cause mitochondrial diseases, and more than 100 pathogenic mutations have been identified in human mtDNA, with a total incidence rate of about 1/5000. In recent years, advances in CRISPR-based base editing technology have enabled accurate editing of nuclear genes. However, it remains a challenge to achieve precise base editing on mtDNA due to the difficulty of guide RNA in the CRISPR system passing through the mitochondrial double-membrane. In 2020, David R. Liu's group at Harvard University reported a double-stranded DNA deaminase DddA from Burkholderia cenocepacia, which was fused with the programmable transcription activator-like effector (TALE) and uracil glycosylase inhibitor (UGI) to develop DddA-derived cytosine base editors (DdCBEs). Using DdCBEs, they were able to achieve specific and efficient C?G to T?A conversion on mtDNA for the first time. In this review, we summarize the recent progress of mitochondrial base editing technology based on DddA and prospect its future application prospects. The information presented may facilitate interested researchers to grasp the principles of mitochondrial base editing, to use relevant base editors in their own studies, or to optimize mitochondrial base editors in the future.
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A Review of CRISPR Tools for Treating Usher Syndrome: Applicability, Safety, Efficiency, and In Vivo Delivery.
This review considers research into the treatment of Usher syndrome, a deaf-blindness syndrome inherited in an autosomal recessive manner. Usher syndrome mutations are markedly heterogeneous, involving many different genes, and research grants are limited due to minimal patient populations. Furthermore, gene augmentation therapies are impossible in all but three Usher syndromes as the cDNA sequence exceeds the 4.7 kb AAV packaging limit. It is, therefore, vital to focus research efforts on alternative tools with the broadest applicability. The CRISPR field took off in recent years following the discovery of the DNA editing activity of Cas9 in 2012. New generations of CRISPR tools have succeeded the original CRISPR/Cas9 model to enable more sophisticated genomic amendments such as epigenetic modification and precise sequence alterations. This review will evaluate the most popular CRISPR tools to date: CRISPR/Cas9, base editing, and prime editing. It will consider these tools in terms of applicability (in relation to the ten most prevalent USH2A mutations), safety, efficiency, and in vivo delivery potential with the intention of guiding future research investment.
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Protocol for permanent gene repression by CRISPR-adenine base editing of promoter CCAAT motifs.
Here, we present a protocol to achieve permanent downregulation of gene expression by editing the CCAAT box in promoter regions using CRISPR-adenine base editors (ABEs). We outline steps for guide RNA (gRNA) design, transfection, genomic DNA extraction, Sanger sequencing, and gene expression quantification. The protocol is optimized for mammalian cell lines (e.g., NIH3T3). It allows for precise disruption of transcription factor binding site without double-strand breaks and offers a novel alternative to RNAi or CRISPR interference (CRISPRi). For complete details on the use and execution of this protocol, please refer to Daliri et al.1.
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Increasing the Targeting Scope of CRISPR Base Editing System Beyond NGG.
Genome editing provides a new therapeutic strategy to cure genetic diseases. The recently developed CRISPR-Cas9 base editing technology has shown great potential to repair the majority of pathogenic point mutations in the patient's DNA precisely. Base editor is the fusion of a Cas9 nickase with a base-modifying enzyme that can change a nucleotide on a single strand of DNA without generating double-stranded DNA breaks. However, a major limitation in applying such a system is the prerequisite of a protospacer adjacent motif sequence at the desired position relative to the target site. Progress has been made to increase the targeting scope of base editors by engineering SpCas9 protein variants, establishing systems with broadened editing windows, characterizing new SpCas9 orthologs, and developing prime editing technology. In this review, we discuss recent progress in the development of CRISPR base editing, focusing on its targeting scope, and we provide a workflow for selecting a suitable base editor based on the target nucleotide sequences.
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Genome editing, a superior therapy for inherited retinal diseases.
Gene augmentation and genome editing are promising strategies for the treatment of monogenic inherited retinal diseases. Although gene augmentation treatments are commercially available for inherited retinal diseases, there are many shortcomings that need to be addressed, like progressive retinal degeneration and diminishing efficacy over time. Innovative CRISPR-Cas9-based genome editing technologies have broadened the proportion of treatable genetic disorders and can greatly improve or complement treatment outcomes from gene augmentation. Progress in this relatively new field involves the development of therapeutics including gene disruption, ablate-and-replace strategies, and precision gene correction techniques, such as base editing and prime editing. By making direct edits to endogenous DNA, genome editing theoretically guarantees permanent gene correction and long-lasting treatment effects. Improvements to delivery modalities aimed at limiting persistent gene editor activity have displayed an improved safety profile and minimal off-target editing. Continued progress to advance precise gene correction and associated delivery strategies will establish genome editing as the preferred treatment for genetic retinal disorders. This commentary describes the applications, strengths, and drawbacks of conventional gene augmentation approaches, recent advances in precise genome editing in the retina, and promising preclinical strategies to facilitate the use of robust genome editing therapies in human patients.
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Rapid Vector Construction and Assessment of BE3 and Target-AID C to T Base Editing Systems in Rice Protoplasts.
CRISPR-Cas9 has revolutionized the field of genome engineering. Base editing, a new genome editing strategy, was recently developed to engineer nucleotide substitutions. DNA base editing systems use a catalytically impared Cas nuclease together with a nucleobase deaminase enzyme to specifically introduce point mutations without generating double-stranded breaks, which provide huge potential in crop improvement. Here, we describe fast and efficient preparation of user-friendly C to T base editors, BE3, and Target-AID. Presented are detailed protocols for T-DNA vector preparation with BE3 or modified Target-AID base editor based on Gateway assembly and efficiency assessment of base editing through a rice protoplast transient expression system.
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The emerging impact of CRISPR and gene editing on global crop improvement.
The advent of CRISPR-based genome editing has revolutionized crop improvement, offering unprecedented precision and efficiency in modifying key agronomic traits. This review comprehensively examines the mechanisms, applications, and future potential of CRISPR technology in enhancing global crop production. CRISPR-Cas systems, originally identified as adaptive immune mechanisms in bacteria and archaea, have been repurposed for targeted genome editing in plants. The CRISPR-Cas9 system, in particular, has emerged as a powerful tool for introducing site-specific double-strand breaks, enabling precise genetic modifications. The three-stage process of adaptation, expression, and interference underlies the CRISPR mechanism, with guide RNAs directing Cas endonucleases to specific genomic loci. Advances in CRISPR technology have expanded its applications beyond gene knockouts, encompassing base editing, prime editing, and epigenome editing. These innovations have facilitated the development of crops with enhanced yield, stress tolerance, disease resistance, nutritional content, and post-harvest quality. However, challenges related to off-target effects, regulatory hurdles, ethical concerns, and public acceptance must be addressed to fully harness the potential of CRISPR in agriculture. Integration of CRISPR with other cutting-edge technologies, such as synthetic biology, artificial intelligence, and high-throughput phenotyping, holds immense promise for accelerating crop improvement efforts. As research continues to refine CRISPR tools and expand their applicability across diverse plant species, this transformative technology is poised to play a pivotal role in shaping a sustainable, resilient, and productive global food system for future generations.
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Base editing the synapse: Modeling a complex neurological disorder in non-human primates.
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CRISPR-based genome editing in human embryos: a review of efficiency, safety, and ethical implications.
Programmable gene editing tools, particularly CRISPR/Cas9 and its advanced derivatives (base and prime editors), have revolutionized biomedical research and offer unprecedented potential for studying human embryogenesis and correcting monogenic diseases. This review systematically examines the evolution and challenges of these technologies in human embryos and mammalian models. We trace key methodological advancements, from initial studies hampered by low efficiency and mosaicism to refined strategies like RNP delivery and base editing that improved precision. A critical shift occurred with the discovery that CRISPR/Cas9 can cause severe on-target damage, such as large deletions and chromosomal loss, redirecting the field's focus toward safety. We present a comparative analysis of editing efficiencies across species (human, mouse, primate, pig, cow, and rabbit) and tools (Cas9, BEs, and PEs), consistently demonstrating the superiority of RNP for precise editing. Fundamental barriers to clinical translation are discussed, including the trade-off between efficiency and mosaicism, persistent off-target effects, and profound ethical concerns. The review concludes that while somatic gene therapy advances rapidly, heritable genome editing remains premature due to unresolved risks. Future progress depends on developing safer editors, understanding on-target consequences, and adhering to rigorous ethical standards.
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Reduction in activating transcription factor 4 promotes carbon tetrachloride and lipopolysaccharide/D‑galactosamine‑mediated liver injury in mice.
Although activating transcription factor 4 (ATF4) is involved in the regulation of numerous biological functions, whether ATF4 has a direct role in liver injury is unknown. The aim of the present study was to investigate the role of ATF4 in liver injury using mouse models. The results revealed that ATF4 protein is expressed markedly higher in the mouse liver when in comparison with other tissues. Notably, tunicamycin treatment, an endoplasmic reticulum (ER) stress inducer, induced the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), but decreased ATF4 protein levels in the mouse liver. This suggested an unconventional regulation pattern of ATF4 protein not associated with ER stress or eIF2α. In addition, it was also observed that the liver levels of ATF4 protein were significantly reduced upon chronic liver injury induced by carbon tetrachloride (CCl4). ATF4 protein was also decreased in acute liver injury induced by lipopolysaccharide (LPS) plus D‑galactosamine (D‑GalN). Furthermore, the results revealed that knockdown of ATF4 by injecting ATF4‑targeting Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‑CRISPR associated protein 9 plasmids exacerbated CCl4 and LPS/D‑GalN‑induced liver injury as demonstrated by elevated serum aspartate transaminase and alanine aminotransferase levels. ATF4 suppression also enhanced CCl4 and LPS/D‑GalN mediated c‑Jun N‑terminal kinase activation. By contrast, ATF4 overexpression alleviated CCl4 and LPS/D‑GalN‑induced liver injury. Taken together, these observations suggested that ATF4 may serve a protective role in the mouse liver.
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