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Developing a base-editing system to expand the carbon source utilization spectra of Shewanella oneidensis MR-1 for enhanced pollutant degradation.
Shewanella oneidensis MR-1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR-1 as carbon sources, resulting in its limited applications. In this study, a rapid, highly efficient, and easily manipulated base-editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR-1. The C-to-T conversion of suitable C within the base-editing window could be readily and efficiently achieved by the pCBEso system without requiring double-strand break or repair templates. Moreover, double-locus simultaneous editing was successfully accomplished with an efficiency of 87.5%. With this tool, the key genes involving in N-acetylglucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR-1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild-type when glucose or GlcNAc was used as the sole carbon source. Such a base-editing system could be readily applied to other EEB. This study not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR-1 but also accelerates the robust construction of engineered strains for environmental bioremediation.
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Advances in multiplex precision genome editing in eukaryotic and prokaryotic systems.
Multiplex genome editing (MGE) enables coordinated modification of multiple genomic loci and is foundational for engineering complex biological traits. Traditional CRISPR-Cas nuclease-based strategies rely on DNA double-strand breaks (DSBs), which limit precision and pose scaling challenges for incorporating simultaneous edits across different loci. Recent advances in genome editing technologies that operate without generating DSBs have expanded the accuracy and feasibility of multiplexed genomic manipulation. This review focuses on emerging strategies for precise MGE, including base editing, prime editing, and related genome rewriting platforms. We highlight key engineering principles that impact the success of scalable multiplexing, including the choice of editing platform, edit size, and guide RNA architecture, and discuss applications across mammalian, plant, fungal, and bacterial systems. Together, these technologies establish MGE as a versatile framework for precise multigene control in biotechnology and agriculture.
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Genome scale analysis of pathogenic variants targetable for single base editing.
BACKGROUND: Single nucleotide variants account for approximately 90% of all known pathogenic variants responsible for human diseases. Recently discovered CRISPR/Cas9 base editors can correct individual nucleotides without cutting DNA and inducing double-stranded breaks. We aimed to find all possible pathogenic variants which can be efficiently targeted by any of the currently described base editors and to present them for further selection and development of targeted therapies. METHODS: ClinVar database (GRCh37_clinvar_20171203) was used to search and select mutations available for current single-base editing systems. We included only pathogenic and likely pathogenic variants for further analysis. For every potentially editable mutation we checked the presence of PAM. If a PAM was found, we analyzed the sequence to find possibility to edit only one nucleotide without changing neighboring nucleotides. The code of the script to search Clinvar database and to analyze the sequences was written in R and is available in the appendix. RESULTS: We analyzed 21 editing system currently reported in 9 publications. Every system has different working characteristics such as the editing window and PAM sequence. C > T base editors can precisely target 3196 mutations (46% of all pathogenic T > C variants), and A > G editors - 6900 mutations (34% of all pathogenic G > A variants). CONCLUSIONS: Protein engineering helps to develop new enzymes with a narrower window of base editors as well as using new Cas9 enzymes with different PAM sequences. But, even now the list of mutations which can be targeted with currently available systems is huge enough to choose and develop new targeted therapies.
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Phage peptides mediate precision base editing with focused targeting window.
Base editors (BEs) are genome engineering tools that can generate nucleotide substitutions without introducing double-stranded breaks (DSBs). A variety of strategies have been developed to improve the targeting scope and window of BEs. In a previous study, we found that a bacteriophage-derived peptide, referred to as G8PPD, could improve the specificity of Cas9 nuclease. Herein, we investigate the applicability of G8PPD as molecular modulators of BEs. We show that G8PPD can improve cytidine base editor (CBEs) and adenine base editor (ABE) to more focused targeting windows. Notably, in a cell-based disease model, G8PPD increases the percentage of perfectly edited gene alleles by BEs from less than 4% to more than 38% of the whole population. In addition, G8PPD can improve the targeting scope of BE in mouse embryos. In summary, our study presents the peptidyl modulators that can improve BEs for precision base editing.
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Recent preclinical and clinical advances in gene therapy for hereditary hearing loss.
Hereditary hearing loss is a genetically heterogeneous condition that affects millions of people worldwide and has limited curative treatment options. Recent advancements in gene therapy have opened promising avenues for correcting the underlying genetic defects in the inner ear. This review summarizes the key developments in vector platforms, delivery strategies, target genes, preclinical models, and clinical trials relevant to both gene supplementation and gene editing approaches, as well as future directions. Adeno-associated virus vectors have emerged as the leading platform for inner ear gene transfer, owing to their safety and efficacy. Clinical programs, such as those targeting OTOF variants, are currently underway and are supported by robust preclinical data. Additionally, genome editing technologies, including CRISPR/Cas9-mediated nonhomologous end joining, base editing, and prime editing, offer variant-specific therapeutic potential. Despite these advances, challenges remain in expanding the therapeutic window, ensuring long-term safety, and establishing ethical and regulatory frameworks for their use.
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Generation of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in Arabidopsis thaliana.
Towards precise genome editing, base editors have been developed by fusing catalytically compromised Cas9 with deaminase components, mediating C-to-T (cytosine base editors) or A-to-G (adenine base editors) transition. We developed a set of vectors consisting of a 5'-NG-3' PAM-recognizing variant of SpCas9 with adenosine deaminases TadA7.10 or TadA8e. Using a phenotype-based screen in Arabidopsis thaliana targeting multiple PDS3 intron splice sites, we achieved up to 81% somatic A-to-G editing in primary transformants at a splice acceptor site with NGG PAM, while 35% was achieved for the same target adenine with NGA PAM. Among tested vectors, pECNUS4 (Addgene #184887), carrying TadA8e, showed the highest adenine base editor (ABE) efficiency. With pECNUS4, we recreated a naturally occurring allele of DANGEROUS MIX3 (DM3) in two generations, transgene-free, for NGC PAM. We also simultaneously base-edited four redundant DM1/SSI4 homologs, encoding nucleotide-binding leucine-rich repeat (NLR) proteins, using a single gRNA with NGA PAM targeting the conserved yet functionally crucial P-loop motif of NLR proteins. We found fixation of A-to-G in three NLR genes for all three possible adenine sites within base-editing window 3-9, as the edited genes segregate in T2. Multigene targeting succeeded in rescuing the previously reported autoimmune phenotype in two generations. Mediating desired ABE on seven NLR genes simultaneously was successful as well; above 77% editing was achieved in six of the seven possible targets in a T1 plant, with the remaining having a moderately high (32%) editing. ABE application to specifically inactivate functional motifs is anticipated to expedite the discovery of novel roles for proteins. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
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Web-Based Computational Tools for Base Editors.
CRISPR-based base editors are efficient genome editing tools for use in base correction. Currently, there are various versions and types of base editors with different substitution patterns, editing windows, and protospacer adjacent motif (PAM) sequences. For the design of target sequences, consideration of off-target sequences is required. In addition, for assessment of base editing outcomes in bulk populations, the analysis of high-throughput sequencing data is required. Several web browser-based computation programs have been developed for the purpose of target design and NGS data analysis, especially for users with less computational knowledge. In this manuscript, depending on the purpose of each program, we provide an explanation of useful tools including BE-Designer for design of targets and BE-Analyzer for analysis of NGS data that were developed by our group, CRISPResso2 for analysis of NGS data developed by Luca Pinello group, DeepBaseEditor for prediction of target efficiency developed by Hyongbum Henry Kim group, and BE-Hive for prediction of target outcome developed by David Liu group.
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Current Status and Challenges of DNA Base Editing Tools.
CRISPR-mediated DNA base editors, which include cytosine base editors (CBEs) and adenine base editors (ABEs), are promising tools that can induce point mutations at desired sites in a targeted manner to correct or disrupt gene expression. Their high editing efficiency, coupled with their ability to generate a targeted mutation without generating a DNA double-strand break (DSB) or requiring a donor DNA template, suggests that DNA base editors will be useful for treating genetic diseases, among other applications. However, this hope has recently been challenged by the discovery of DNA base editor shortcomings, including off-target DNA editing, the generation of bystander mutations, and promiscuous deamination effects in both DNA and RNA, which arise from the main DNA base editor constituents, a Cas nuclease variant and a deaminase. In this review, we summarize information about the DNA base editors that have been developed to date, introduce their associated potential challenges, and describe current efforts to minimize or mitigate those issues of DNA base editors.
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In vivo base editing reduces liver cysts in autosomal dominant polycystic kidney disease.
Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent genetic kidney disorder, affecting over 10 million individuals worldwide. Cystic expansion typically progresses to kidney failure and also involves the liver with limited treatment options. Pathogenic variants in PKD1 or PKD2 account for 85%-90% of cases. Genetic re-expression of Pkd1 or Pkd2 has been shown to partially reverse key characteristics of the disease phenotype in mice. Despite advancements in understanding the genetic basis, it remains unclear whether correcting pathogenic variants can effectively prevent, modify, or reverse the disease. Additionally, the feasibility of genome editing as a treatment remains largely unexplored. In this study, we employed CRISPR base editing to correct representative pathogenic PKD1 variants selected from a patient cohort, achieving precise and efficient editing in vitro. Correction of a murine missense variant (c.6646C>T (R2216W)) in primary renal epithelial cells increased polycystin-1 expression and reduced the endoplasmic reticulum stress marker sXBP1. In vivo, base editor delivery to the c.6646C>T (R2216W) knockin mouse enabled correction of the pathogenic variant, resulting in a significant reduction in liver cysts. These findings provide the first evidence that genome editing may ameliorate key features of ADPKD, opening promising therapeutic perspectives for affected patients and their families.
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Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion.
Nucleotide base editors in plants have been limited to conversion of cytosine to thymine. Here, we describe a new plant adenine base editor based on an evolved tRNA adenosine deaminase fused to the nickase CRISPR/Cas9, enabling A•T to G•C conversion at frequencies up to 7.5% in protoplasts and 59.1% in regenerated rice and wheat plants. An endogenous gene is also successfully modified through introducing a gain-of-function point mutation to directly produce an herbicide-tolerant rice plant. With this new adenine base editing system, it is now possible to precisely edit all base pairs, thus expanding the toolset for precise editing in plants.
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