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Adenine base editors catalyze cytosine conversions in human cells.
Adenine base editors comprise an adenosine deaminase, evolved in vitro, and a Cas9 nickase. Here, we show that in addition to converting adenine to guanine, adenine base editors also convert cytosine to guanine or thymine in a narrow editing window (positions 5-7) and in a confined TC*N sequence context. Adenine base editor-induced cytosine substitutions occur independently of adenosine conversions with an efficiency of up to 11.2% and reduce the number of suitable targeting sites for high-specificity base editing.
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Using Gene Editing Approaches to Fine-Tune the Immune System.
Genome editing technologies not only provide unprecedented opportunities to study basic cellular system functionality but also improve the outcomes of several clinical applications. In this review, we analyze various gene editing techniques used to fine-tune immune systems from a basic research and clinical perspective. We discuss recent advances in the development of programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases. We also discuss the use of programmable nucleases and their derivative reagents such as base editing tools to engineer immune cells via gene disruption, insertion, and rewriting of T cells and other immune components, such natural killers (NKs) and hematopoietic stem and progenitor cells (HSPCs). In addition, with regard to chimeric antigen receptors (CARs), we describe how different gene editing tools enable healthy donor cells to be used in CAR T therapy instead of autologous cells without risking graft-versus-host disease or rejection, leading to reduced adoptive cell therapy costs and instant treatment availability for patients. We pay particular attention to the delivery of therapeutic transgenes, such as CARs, to endogenous loci which prevents collateral damage and increases therapeutic effectiveness. Finally, we review creative innovations, including immune system repurposing, that facilitate safe and efficient genome surgery within the framework of clinical cancer immunotherapies.
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Design of highly functional genome editors by modelling CRISPR-Cas sequences.
Gene editing has the potential to solve fundamental challenges in agriculture, biotechnology and human health. CRISPR-based gene editors derived from microorganisms, although powerful, often show notable functional tradeoffs when ported into non-native environments, such as human cells1. Artificial-intelligence-enabled design provides a powerful alternative with the potential to bypass evolutionary constraints and generate editors with optimal properties. Here, using large language models2 trained on biological diversity at scale, we demonstrate successful precision editing of the human genome with a programmable gene editor designed with artificial intelligence. To achieve this goal, we curated a dataset of more than 1 million CRISPR operons through systematic mining of 26 terabases of assembled genomes and metagenomes. We demonstrate the capacity of our models by generating 4.8× the number of protein clusters across CRISPR-Cas families found in nature and tailoring single-guide RNA sequences for Cas9-like effector proteins. Several of the generated gene editors show comparable or improved activity and specificity relative to SpCas9, the prototypical gene editing effector, while being 400 mutations away in sequence. Finally, we demonstrate that an artificial-intelligence-generated gene editor, denoted as OpenCRISPR-1, exhibits compatibility with base editing. We release OpenCRISPR-1 to facilitate broad, ethical use across research and commercial applications.
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Identification of optimal adenine and cytosine base editors for genome editing in Arabidopsis and soybean.
Base editors, including adenine base editors (ABEs) and cytosine base editors (CBEs), are widely used in numerous organisms to introduce site-specific sequence modifications in genomic DNA without causing double-strand breaks (DSBs). However, these editors exhibit low editing efficiencies, particularly in dicot plants, thereby limiting their application in dicot plant genome engineering. In this study, we assessed the editing efficiencies of various base editors to identify those optimal for base editing in dicot plants. We discovered that ABE8e, an ABE variant, demonstrated superior A-to-G base editing efficiency within A5-A8 windows, and A3A/Y130F-V04, a CBE variant, exhibited the highest C-to-T base editing efficiency within C4-C15 windows in both Arabidopsis and soybean protoplasts. Overall, we recommend these two base editors as prime choices for efficient genome engineering in a range of crop plants. [BMB Reports 2025; 58(7): 288-292].
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Activity-based selection for enhanced base editor mutational scanning.
Base editing is a CRISPR-based technology that enables high-throughput, nucleotide-level functional interrogation of the genome that is essential for understanding the genetic basis of human disease and informing therapeutic development. Base editing screens have emerged as a powerful experimental approach, yet significant cell-to-cell variability in editing efficiency introduces noise that may obscure meaningful results. Here we develop a co-selection method that enriches for cells with high base editing activity, substantially increasing editing efficiency at a target locus. We evaluate this activity-based selection method against a traditional screening approach by tiling guide RNAs across TP53, demonstrating its enhanced capacity to pinpoint specific mutations and protein regions of functional importance. We anticipate that this modular selection method will enhance the resolution of base editing screens across many applications.
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A side-by-side comparison of variant function measurements using deep mutational scanning and base editing.
Variant annotation is a crucial objective in mammalian functional genomics. Deep mutational scanning (DMS) using saturation libraries of complementary DNAs (cDNAs) is a well-established method for annotating human gene variants, but CRISPR base editing (BE) is emerging as an alternative. However, questions remain about how well high-throughput BE measurements can annotate variant function and the extent of downstream experimental validation required. This study is the first direct comparison of cDNA DMS and BE in the same lab and cell line. We focus on how well short guide RNA (sgRNA) depletion or enrichment is explained by the predicted edits within the editing "window" defined by the sgRNA. The most likely predicted edits enhance the agreement between a "gold standard" DMS dataset and a BE screen. A simple filter for sgRNAs making single edits in their window could sufficiently annotate a large proportion of variants directly from sgRNA sequencing of large pools. When multi-edit guides are unavoidable, directly measuring edits in medium-sized validation pools can recover high-quality variant annotation data. Our data show a surprisingly high degree of correlation between base editor data and gold standard DMS. We suggest that the main variable measured in base editor screens is the desired base edits.
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Highly efficient base editing with expanded targeting scope using SpCas9-NG in rabbits.
Base editors, composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable efficient C-to-T or A-to-G conversion in various organisms. However, the NGG protospacer adjacent motif (PAM) requirement of Streptococcus pyogenes Cas9 (SpCas9) substantially limits the target sites suitable for base editing. Quite recently, a new engineered SpCas9-NG variant, which can recognize minimal NG PAMs more efficiently than the present xCas9 variant. Here, we investigated the efficiency and PAM compatibility of SpCas9-NG-assisted cytidine base editors (CBEs) and adenine base editors (ABEs) in rabbits. In this study, we showed that NG-BE4max and NG-ABEmax systems can achieve a targeted mutation efficiency of 75%-100% and 80%-100% with excellent PAM compatibility of NGN PAMs in rabbit embryos, respectively. In addition, both base editors were successfully applied to create new rabbit models with precise point mutations, demonstrating their high efficiency and expanded genome-targeting scope in rabbits. Meanwhile, NG-ABEmax can be used to precisely mimic human Hoxc13 p.Q271R missense mutation in Founder (F0) rabbits, which is arduous for conventional ABEs to achieve due to a NGA PAM requirement. Collectively, NG-BE4max and NG-ABEmax systems provide promising tools to perform efficient base editing with expanded targeting scope in rabbits and enhances its capacity to model human diseases.
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CRISPR/Cas9 gene editing for genodermatoses: progress and perspectives.
Genodermatoses constitute a clinically heterogeneous group of devastating genetic skin disorders. Currently, therapy options are largely limited to symptomatic treatments and although significant advances have been made in ex vivo gene therapy strategies, various limitations remain. However, the recent technical transformation of the genome editing field promises to overcome the hurdles associated with conventional gene addition approaches. In this review, we discuss the need for developing novel treatments and describe the current status of gene editing for genodermatoses, focusing on a severe blistering disease called epidermolysis bullosa (EB), for which significant progress has been made. Initial research utilized engineered nucleases such as transcription activator-like effector nucleases and meganucleases. However, over the last few years, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) have upstaged older generation gene editing tools. We examine different strategies for CRISPR/Cas9 application that can be employed depending on the type and position of the mutation as well as the mode of its inheritance. Promising developments in the field of base editing opens new avenues for precise correction of single base substitutions, common in EB and other genodermatoses. We also address the potential limitations and challenges such as safety concerns and delivery efficiency. This review gives an insight into the future of gene editing technologies for genodermatoses.
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Targeting the hallmarks of ageing: Pharmacological challenges and breakthroughs in CRISPR delivery systems and future prospects.
CRISPR has emerged as a next-generation gene-editing tool with the potential to target the molecular pathways associated with ageing and related disorders. It functions through RNA-guided Cas nucleases, directing DNA cleavage and utilizing the native DNA repair machinery for genetic manipulations. Advances in CRISPR technology have significantly enhanced the precision and flexibility of techniques for genome editing. The enzyme Cas9's ability to cut DNA at exact site has revolutionized genome editing by enabling accurate modifications within living eukaryotic cells. This review critically examines recent developments in CRISPR-based technologies, including Cas9, Cas12, base editing, prime editing, and CRISPR-mediated gene regulation. It highlights their rising applications in ageing research, with more emphasis on neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The review also discusses the major pharmacological and translational challenges that currently limit clinical applications, including inefficient tissue-specific delivery, off-target genome editing, immunogenicity, manufacturing complexity, and long-term safety concerns. Also, recent progress in both, viral and non-viral delivery methods are critically evaluated, including adeno-associated viruses, lentivirus vectors, lipid nanoparticles, gold nanoparticles, exosomes, electroporation, and microinjection, is thoroughly discussed to highlight their therapeutic potential and translational limitations. Current studies indicate that CRISPR-based approaches have preclinical potential for targeting important hallmarks of ageing, particularly genomic instability, telomere attrition, and mitochondrial dysfunction. Other hallmarks of ageing, such as stem cell exhaustion, epigenetic modifications, and microbiome changes, are at earlier stages of development. Overall, this review describes future strategies for developing safe, precise, and clinically translatable CRISPR-based treatments to promote healthy ageing.
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Fast and Efficient Generation of Isogenic Induced Pluripotent Stem Cell Lines Using Adenine Base Editing.
Isogenic induced pluripotent stem cell (iPSC) lines are currently mostly created by homology directed repair evoked by a double-strand break (DSB) generated by CRISPR-Cas9. However, this process is in general lengthy and inefficient. This problem can be overcome, specifically for correction or insertion of transition mutations, by using base editing (BE). BE does not require DSB formation, hence avoiding creation of genomic off-target breaks and insertions and deletions, and as it is highly efficient, it also does not require integration of selection cassettes in the genome to enrich for edited cells. BE has been successfully used in many cell types as well as in some in vivo settings to correct or insert mutations, but very few studies have reported generation of isogenic iPSC lines using BE. Here, we describe a simple and fast workflow to generate isogenic iPSCs efficiently with a compound heterozygous or a homozygous Wolfram syndrome 1 (WFS1) mutation using adenine BE, without the need to include a genomic selection cassette and without off-target modifications. We demonstrated that correctly base-edited clones can be generated by screening only five cell clones in less than a month, provided that the mutation is positioned in a correct place with regards to the protospacer adjacent motif sequence and no putative bystander bases exist.
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