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Efficient A-to-C base editing with high specificity.
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Parallel engineering and activity profiling of a base editor system.
Selecting the most suitable existing base editors and engineering new variants for installing specific base conversions with maximal efficiency and minimal undesired edits are pivotal for precise genome editing applications. Here, we present a platform for creating and analyzing a library of engineered base editor variants to enable head-to-head evaluation of their editing performance at scale. Our comprehensive comparison provides quantitative measures on each variant's editing efficiency, purity, motif preference, and bias in generating single and multiple base conversions, while uncovering undesired higher indel generation rate and noncanonical base conversion for some of the existing base editors. In addition to engineering the base editor protein, we further applied this platform to investigate a hitherto underexplored engineering route and created guide RNA scaffold variants that augment the editor's base-editing activity. With the unknown performance and compatibility of the growing number of engineered parts including deaminase, CRISPR-Cas enzyme, and guide RNA scaffold variants for assembling the expanding collection of base editor systems, our platform addresses the unmet need for an unbiased, scalable method to benchmark their editing outcomes and accelerate the engineering of next-generation precise genome editors.
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Base editing in rice: current progress, advances, limitations, and future perspectives.
Base editing is one of the promising genome editing tools for generating single-nucleotide changes in rice genome. Rice (Oryza sativa L.) is an important staple food crop, feeding half of the population around the globe. Developing new rice varieties with desirable agronomic traits is necessary for sustaining global food security. The use of genome editing technologies for developing rice varieties is pre-requisite in the present scenario. Among the genome editing technologies developed for rice crop improvement, base editing technology has emerged as an efficient and reliable tool for precise genome editing in rice plants. Base editing technology utilizes either adenosine or cytidine base editor for precise editing at the target region. A base editor (adenosine or cytidine) is a fusion of catalytically inactive CRISPR/Cas9 domain and adenosine or cytidine deaminase domain. In this review, authors have discussed the different adenine and cytosine base editors developed so far for precise genome editing of rice via base editing technology. We address the current progress, advances, limitations, as well as future perspectives of the base editing technology for rice crop improvement.
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MACBETH: Multiplex automated Corynebacterium glutamicum base editing method.
CRISPR/Cas9 or Cpf1-introduced double strand break dramatically decreases bacterial cell survival rate, which hampers multiplex genome editing in bacteria. In addition, the requirement of a foreign DNA template for each target locus is labor demanding and may encounter more GMO related regulatory hurdle in industrial applications. Herein, we developed a multiplex automated Corynebacterium glutamicum base editing method (MACBETH) using CRISPR/Cas9 and activation-induced cytidine deaminase (AID), without foreign DNA templates, achieving single-, double-, and triple-locus editing with efficiencies up to 100%, 87.2% and 23.3%, respectively. In addition, MACBETH was applied to generate a combinatorial gene inactivation library for improving glutamate production, and pyk&ldhA double inactivation strain was found to improve glutamate production by 3-fold. Finally, MACBETH was automated with an integrated robotic system, which would enable us to generate thousands of rationally engineered strains per month for metabolic engineering of C. glutamicum. As a proof of concept demonstration, the automation platform was used to construct an arrayed genome-scale gene inactivation library of 94 transcription factors with 100% success rate. Therefore, MACBETH would be a powerful tool for multiplex and automated bacterial genome editing in future studies and industrial applications.
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Base Editors for Engineering Industrial Microorganisms: Types, Applications, and Future Perspectives.
Base editing encompasses technologies that enable the direct conversion of one nucleotide base into another, typically by employing deaminases fused to programmable DNA- or RNA-binding scaffolds such as CRISPR-dCas9/nCas9. Deaminase-free editing strategies are also emerging as promising alternatives. Unlike conventional CRISPR-Cas9 systems that rely on DNA double-strand breaks (DSBs) and subsequent homology-directed repair or non-homologous end joining (NHEJ), base editors operate independently of these pathways, thus enabling continuous in vivo genome evolution and phenotypic diversification. While previous reviews have summarized base editing in plants and animals, the present review focuses specifically on base editors tailored for industrial microorganisms. We provide a comprehensive overview of deaminase-dependent and glycosylase-dependent base editors, emphasizing their applications in sculpting microbial genomes, redirecting metabolic flux, and enhancing stress tolerance. In addition, we summarize recent advances in in situ bacterial base editing, an emerging frontier for industrial strain improvement. Finally, we dissect the current limitations of these tools and propose actionable strategies to enhance editing performance and broaden their applicability. By integrating these perspectives, this review aims to guide future development and deployment of base editing technologies in both model and non-model industrial microorganisms, ultimately advancing their roles in biomanufacturing, biomonitoring, and beyond.
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[Gene editing is changing the treatment of hereditary diseases].
Gene editing is a novel technology within gene therapy, which changes sequences in chromosomal DNA with precision. Even if there are alternative strategies, the Nobel Prize-winning CRISPR/Cas technology has become the dominating principle. During recent years base editing and prime editing, permitting editing without DNA double-strand breaks, have been developed. The first clinical gene editing results were reported in 2021; since then many patients have been treated, and recently the first treatment was approved as a novel therapy in the UK and later in USA and in EU. This update describes various aspects including methodological developments and safety.
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High-efficiency base editing for nuclear and mitochondrial DNA with an optimized DYW-like deaminase.
CRISPR-based cytosine base editors enable precise genome editing without inducing double-stranded DNA breaks yet traditionally depend on a limited selection of deaminases from the APOBEC/AID or TadA families. Here, we present SsCBE, a CRISPR-based cytosine base editor utilizing SsdAtox, a DYW-like deaminase derived from the toxin of Pseudomonas syringae. Strategic engineering of SsdAtox has led to remarkable improvements in the base editing efficiency (by up to 8.4-fold) and specificity for SsCBE, while concurrently reducing cytotoxicity. Exhibiting exceptional versatility, SsCBE was delivered and efficiently applied using diverse delivery methods, including engineered virus-like particles. Its application has enabled targeted cytosine base editing in mouse zygotes and pioneering edits in mitochondrial DNA. SsCBE expands the genome editing toolbox by introducing a distinct deaminase scaffold with broad utility for both basic research and potential therapeutic applications.
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Orthogonal CRISPR systems for targeted integration and multiplex base editing enable nonviral engineering of allogeneic CAR-T cells.
Multiple genomic modifications, including targeted transgene integrations and knockouts, may be required to develop potent, allogeneic chimeric antigen receptor (CAR)-T cell therapies. Conventional CRISPR-Cas systems generate double-strand breaks (DSBs) associated with genomic rearrangements and genotoxicities. DSB-free base editing reduces these risks. Here, we facilitate multiplex editing by combining Staphylococcus aureus Cas9 (SaCas9) mRNA base editors for DSB-free knockout of B2M and REGNASE-1 with Streptococcus pyogenes Cas9 nucleases for targeted integration of an anti-CD19 CAR transgene at the T cell receptor α constant locus. Combined, these edits have been reported to generate safer allogeneic CAR-T cells with enhanced activity and persistence. We demonstrate multiplex gene editing in primary human T cells with B2M and REGNASE-1 base editing frequencies reaching 66% and 84%, respectively, while integrating the anti-CD19 CAR transgene in up to 36% or 71% of cells using nonviral single-stranded DNA repair templates or viral vector templates (AAV6), respectively. Importantly, no detrimental effects on CAR-T cell function were observed in vitro or in vivo, and knockout by base editing reduced rates of balanced chromosomal translocations by 210-fold. This orthogonal CRISPR-Cas engineering approach represents a novel and safer strategy for nonviral, multiplexed genetic engineering of CAR-T cells.
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Genome-wide CRISPR screen identifies Menin and SUZ12 as regulators of human developmental timing.
Embryonic development follows a conserved sequence of events across species, yet the pace of development is highly variable and particularly slow in humans. Species-specific developmental timing is largely recapitulated in stem cell models, suggesting a cell-intrinsic clock. Here we use directed differentiation of human embryonic stem cells into neuroectoderm to perform a whole-genome CRISPR-Cas9 knockout screen and show that the epigenetic factors Menin and SUZ12 modulate the speed of PAX6 expression during neural differentiation. Genetic and pharmacological loss-of-function of Menin or SUZ12 accelerate cell fate acquisition by shifting the balance of H3K4me3 and H3K27me3 at bivalent promoters, thereby priming key developmental genes for faster activation upon differentiation. We further reveal a synergistic interaction of Menin and SUZ12 in modulating differentiation speed. The acceleration effects were observed in definitive endoderm, cardiomyocyte and neuronal differentiation paradigms, pointing to chromatin bivalency as a general driver of timing across germ layers and developmental stages.
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High-Throughput Gene Mutagenesis Screening Using Base Editing.
Base editing is a CRISPR-Cas9 genome engineering tool that allows programmable mutagenesis without the creation of double-stranded breaks. Here, we describe the design and execution of large-scale base editing screens using the Target-AID base editor in yeast. Using this approach, thousands of sites can be mutated simultaneously. The effects of these mutations on fitness can be measured using a pooled growth competition assay followed by DNA sequencing of gRNAs as barcodes.
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