Introduction
The field of genome editing has undergone a transformative journey, fueled by seminal discoveries in molecular biology. Within the dynamic landscape of genome editing, DNA base editors have emerged as powerful tools for orchestrating precise changes at the nucleotide level. Base editors, which are a subset of genome editing tools, allow modification of individual DNA nucleotides without causing double-strand breaks. The DNA base editors permit the editing of genetic information, thereby opening new avenues for therapeutic interventions and mechanistic exploration. However, despite the promise of base editors, their off-target activity, which entails non-specific DNA editing, poses a critical limitation for their therapeutic use. In genome editing, precision is essential since a single non-specific genome editing event can have far-reaching consequences, such as frame-shift mutations. Frame-shift mutations, which alter the open reading frame of genes, often result from indels introduced during genome editing. These mutations can lead to the production of truncated and/or non-functional proteins, which can result in the loss of normal cell function and cause deleterious biological effects. Therefore, ensuring high specificity and minimizing off-target effects remain critical goals for the therapeutic use of base editors.
The ideal base editor would exhibit robust on-target, and diminished off-target activity. To address these challenges, Zeng et al. (2023) present split adenine base editor (sABE), which is a novel approach designed to address the limitations of existing base editing techniques. sABE utilizes the principles of chemically induced dimerization (CID) in order to infer precise control over the catalytic activity of deoxyadenosine deaminase TadA-8e.
Genome Editing with Split Adenine Base Editors
In genome editing, maximizing on-target and minimizing off-target activity are desirable features. sABE, which are inducible base editors, have the potential to offer this feature by incorporating a rapamycin-based induction for robust on-target editing efficacy akin to its precursor, the original ABE featuring TadA-8e deaminase. The increased precision, characterized by a narrowed activity window on DNA, facilitates an enhanced single-to-double ratio of adenine editing, thereby mitigating unintended genetic alterations, and diminishing off-target effects.
To monitor DNA deaminase activity in vitro, Zeng et al. (2023) developed an innovative fluorescence-based reporter system. Initially, a premature stop codon was introduced within the Enhanced Yellow Fluorescent Protein (EYFP) gene via a C•G (Cytosine to Guanine) to T•A (Thymine to adenine) base pair conversion, which rendered it non-functional. Subsequently, the antisense strand of the EYFP gene was targeted using sABE via a single guide RNA (sgRNA), This precise editing converted the A•T base pair back to the original G•C base pair, thereby restoring the glutamine codon and enabling the expression of full-length, functional EYFP. To achieve inducible control over sABE deaminase, the TadA-8e enzyme was split into two inactive fragments: TadA-8eN and TadA-8eC. These fragments were individually fused to FKBP-rapamycin binding (FRB) and FK506-binding protein 3 (FKBP3) proteins, respectively. Upon exposure to rapamycin, FKBP3 and FRB heterodimerize, bringing the TadA-8eN and TadA-8eC components into proximity. This assembly formed a functional enzymatic unit capable of precise base editing. Furthermore, two versions of split ABE (sABE) were constructed: sABE v1 and sABE v2. These versions were derived from the TadA-8e38 deaminase, with split sites occurring at loop-25 for sABE v1 and loop-74 for sABE v2. For a proof of principle, the co-transfection of HEK293T cells, which is an immortalized cell line derived from human embryonic kidney cells, with plasmids encoding the two sABEs, EYFP, and sgRNA, followed by induction of the sABE activity using rapamycin, successfully activated the EYFP reporter. Subsequent optimization efforts led to the development of sABE v3.22, which exhibited robust induced base editing activity while maintaining minimal non-induced background activity.
Zeng et al. (2023) further demonstrated that the delivery of sABE via dual adeno-associated virus vectors promoted efficient conversion of A•T base pairs to G•C base pairs within the PCSK9 gene in murine hepatic tissue, thus highlighting the use of sABE in vivo. The high on-target and reduced off-target activity of sABE’s offer refined precision and versatility for targeted genome manipulation, thereby promoting the development of innovative therapeutic modalities. Overall, this work demonstrates the successful engineering of a chemically inducible sABE system with precise control over deaminase activity.
Precise Genome Editing by sABE Enhance Their Therapeutic Potential
The development of split editors, such by sABE, represents a breakthrough in precision medicine. Unlike conventional gene-editing methods, which often result in broader genetic alterations due to off-target effects, split editors offer targeted and inducible genome editing with exceptional accuracy, thereby mitigating potential adverse effects. The increased precision offered by sABE’s promises safety in therapeutic applications, prioritizing patient well-being by circumventing unintended genetic modifications. Moreover, split editors exhibit remarkable versatility beyond monogenic diseases to complex conditions influenced by multiple genetic factors, such as diabetes, cancer, and neurodegenerative diseases.
Despite the existing challenges associated with genome editing, ongoing research endeavors focus on optimizing sABE variants, refining delivery vectors, and enhancing safety profiles. These innovations promise precise control over deaminase activity and unprecedented accuracy in addressing health-related concerns, thereby revolutionizing gene therapy and pharmaceuticals development. Overall, this underscores the transformative potential of split editors within the healthcare landscape and personalized medicine.
How Bridge Informatics Can Help
At Bridge Informatics, we are passionate about empowering life science companies with cutting-edge technologies. We believe the split adenine base editor represents a significant advancement in the field of gene editing. Our team of bioinformatics experts can assist your organization in:
- Understanding the implications of this research for your specific drug development programs.
- Developing strategies to incorporate this technology into your research and development pipeline.
- Leveraging our bioinformatics expertise to design and optimize experiments utilizing the split editor.
By embracing innovative solutions like the split adenine base editor, we can collectively accelerate progress towards developing safer and more effective therapies for patients in need. Bridge Informatics’ bioinformaticians are trained bench biologists, so they understand the biological questions driving your computational analysis. Click here to schedule a free introductory call with a member of our team.
Haider M. Hassan, Data Scientist, Bridge Informatics
Haider is one of our premier data scientists. He provides bioinformatic services to clients, including high throughput sequencing, data pre-processing, analysis, and custom pipeline development. Drawing on his rich experience with a variety of high-throughput sequencing technologies, Haider analyzes transcriptional (spatial and single-cell), epigenetic, and genetic landscapes.
Before joining Bridge Informatics, Haider was a Postdoctoral Associate at the London Regional Cancer Centre in Ontario, Canada. During his postdoc, he investigated the epigenetics of late-onset liver cancer using murine and human models. Haider holds a Ph.D. in biochemistry from Western University, where he studied the molecular mechanisms behind oncogenesis. Haider still lives in Ontario and enjoys spending his spare time visiting local parks. If you’re interested in reaching out, please email [email protected] or [email protected]
