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Precision A3G base editors for disease modeling and correction.
Cytosine base editors (CBEs) enable efficient cytosine-to-thymine substitutions at targeted genomic loci without introducing double-stranded breaks. Among CBEs, APOBEC3G BEs (A3G-BEs) preferentially edit the second cytosine within a 5'-CC-3' motif in human cells, reducing potential bystander editing. However, A3G-BEs often unintentionally edit multiple CC motifs within their editing window and are limited by protospacer adjacent motif (PAM) constraints imposed by SpCas9, which restricts their applicability. Here, we engineered A3G-BE variants through linker optimization, rational mutagenesis, and the integration of SpG and SpRY Cas9 effectors with relaxed PAM constraints. These improvements enhanced the precision of single-cytosine editing within CC motifs and broadened the targeting scope to previously inaccessible genomic sites. We then validated the engineered A3G-BE variants by precisely installing and correcting cystic fibrosis-causing mutations in HEK293T cells. When applied to 16HBE14o-human bronchial epithelial cells, precise editing modulated cystic fibrosis transmembrane conductance regulator mRNA levels, protein expression, and channel function, establishing precision A3G-BE variants as powerful tools for modeling and treating cystic fibrosis and other human diseases.
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Prime editing: A gene precision editing tool from inception to present.
Genetic mutations significantly contribute to the onset of diseases, with over half of the cases caused by single-nucleotide mutations. Advances in gene editing technologies have enabled precise editing and correction of mutated genes, offering effective treatment methods for genetic disorders. CRISPR/Cas9, despite its power, poses risks of inducing gene mutations due to DNA double-strand breaks (DSB). The advent of base editing (BE) and prime editing (PE) has mitigated these risks by eliminating the hazards associated with DNA DSBs, allowing for more precise gene editing. This breakthrough lays a solid foundation for the clinical application of gene editing technologies. This review discusses the principles, development, and applications of PE gene editing technology in various genetic mutation-induced diseases.
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Engineering complex phenotypes in Halomonas bluephagenesis TD01 via large-fragment manipulation and multiplex base editing.
Halomonas bluephagenesis is a representative platform strain of next generation industrial biotechnology (NGIB), enabling contamination-resistant open fermentation due to inherent tolerance to high salinity and alkalinity. However, progress in strain development has been constrained by limited genome engineering tools, particularly for large-fragment manipulation and multiplex base editing. Herein, we developed a counterselection marker-based single-plasmid system (pHaloFM) that leverages native homologous recombination to enable sequential insertion of fragments up to 8 kb and deletion of regions up to 50 kb. Additionally, we re-engineered a CRISPR/nCas9-assisted cytidine base editor system (pHaloBE) through host-specific adaptations, achieving multiplex editing of nine target sites. These tools were applied to engineer cellular morphology in one step, and successively construct polyhydroxyalkanoate (PHA) copolymers P34HB and PHBV biosynthetic pathways. This integrated toolkit resolves long-standing genetic manipulation bottlenecks in H. bluephagenesis and provides a systematic framework for engineering complex phenotypes in other non-model organisms.
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CRISPR-Cas9 base editors and their current role in human therapeutics.
BACKGROUND: Consistent progress has been made to create more efficient and useful CRISPR-Cas9-based molecular toolsfor genomic modification. METHODS: This review focuses on recent articles that have employed base editors (BEs) for both clinical and research purposes. RESULTS: CRISPR-Cas9 BEs are a useful system because of their highefficiency and broad applicability to gene correction and disruption. In addition, base editing has beensuggested as a safer approach than other CRISPR-Cas9-based systems, as it limits double-strand breaksduring multiplex gene knockout and does not require a toxic DNA donor molecule for genetic correction. CONCLUSION: As such, numerous industry and academic groups are currently developing base editing strategies withclinical applications in cancer immunotherapy and gene therapy, which this review will discuss, with a focuson current and future applications of in vivo BE delivery.
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Development and validation of optimized lentivirus-like particles for gene editing tool delivery with Gag-Only strategy.
BACKGROUND: The development of gene editing tools such as CRISPR-Cas9 and base editors (BE) is critical for genetic diseases and cancer. Lentivirus-like particles (LVLPs) grows into an auspicious platform for delivering mRNA or ribonucleic proteins (RNPs) due to it integrates the advantage of viral and non-viral vectors. Current LVLP systems predominantly utilize HIV-Gag and Pol proteins. However, the reverse transcriptase and integrase of Pol, pose risks of genomic integration and potential tumorigenesis. Enhancing the safety of VLP system is essential. This study focuses on improving the LVLP to minimize these risks. METHODS: We implemented a Gag-Only strategy, constructing LVLPs with HIV-Gag protein, thereby eliminating the integration risks linked to Pol. By leveraging the interactions between MS2-MCP (MS2 coat protein), PP7 and PP7 BP (PP7 binding protein), and the psi (HIV packaging signal) with HIV-Gag, we encapsulated PAMless andesine base editor (CE-8e-SpRY) mRNA and sgRNA targeting the PD1 start codon (ATG) into the LVLP. Using recombinant lentiviral vector technology, we developed a stable PD1-expressing 293T cell line (PD1-293T) to assess the editing efficiency of LVLP. RESULTS: The psi-LVLP demonstrated effective packaging capabilities, achieving 15% base editing efficiency in 293T cells. By optimizing plasmid ratios, we observed increased CE-8e-SpRY mRNA copy numbers, with 30% base editing efficiency. Additionally, the integration of HDVrz (hepatitis delta virus ribozyme) and psi into sgRNA (HDVrz-psi-LVLP) substantially enhanced sgRNA copy numbers, resulting in approximately 50% base editing efficiency in 293T cells and 20% base editing efficiency in Jurkat cells. Mendelian randomization analyses revealed significant genetic correlations between PD1, B2M, CIITA, and TIGIT genes with various cancer risks. Furthermore, HDVrz-psi-LVLPs targeting the start codons of B2M, CIITA, and TIGIT exhibited high base editing activity in both Jurkat and 293T cells. CONCLUSION: In conclusion, this optimized platform effectively encapsulates CE-8e-SpRY mRNA and sgRNA, achieving high editing efficiencies across multiple genes and cell types. We introduce a safer and more efficient gene editing tool delivery system by constructing LVLPs based on the Gag-Only strategy. Our study presents a promising implication for cancer immunotherapy.
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Engineered nucleocytosolic vehicles for loading of programmable editors.
Advanced gene editing methods have accelerated biomedical discovery and hold great therapeutic promise, but safe and efficient delivery of gene editors remains challenging. In this study, we present a virus-like particle (VLP) system featuring nucleocytosolic shuttling vehicles that retrieve pre-assembled Cas-effectors via aptamer-tagged guide RNAs. This approach ensures preferential loading of fully assembled editor ribonucleoproteins (RNPs) and enhances the efficacy of prime editing, base editing, trans-activators, and nuclease activity coupled to homology-directed repair in multiple immortalized, primary, stem cell, and stem-cell-derived cell types. We also achieve additional protection of inherently unstable prime editing guide RNAs (pegRNAs) by shielding the 3'-exposed end with Csy4/Cas6f, further enhancing editing performance. Furthermore, we identify a minimal set of packaging and budding modules that can serve as a platform for bottom-up engineering of enveloped delivery vehicles. Notably, our system demonstrates superior per-VLP editing efficiency in primary T lymphocytes and two mouse models of inherited retinal disease, highlighting its therapeutic potential.
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Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing.
CRISPR/Cas technologies constitute a powerful tool for genome engineering, yet their use in non-traditional bacteria depends on host factors or exogenous recombinases, which limits both efficiency and throughput. Here we mitigate these practical constraints by developing a widely-applicable genome engineering toolset for Gram-negative bacteria. The challenge is addressed by tailoring a CRISPR base editor that enables single-nucleotide resolution manipulations (C·G → T·A) with >90% efficiency. Furthermore, incorporating Cas6-mediated processing of guide RNAs in a streamlined protocol for plasmid assembly supports multiplex base editing with >85% efficiency. The toolset is adopted to construct and deconstruct complex phenotypes in the soil bacterium Pseudomonas putida. Single-step engineering of an aromatic-compound production phenotype and multi-step deconstruction of the intricate redox metabolism illustrate the versatility of multiplex base editing afforded by our toolbox. Hence, this approach overcomes typical limitations of previous technologies and empowers engineering programs in Gram-negative bacteria that were out of reach thus far.
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BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish.
The ease of use and robustness of genome editing by CRISPR/Cas9 has led to successful use of gene knockout zebrafish for disease modeling. However, it still remains a challenge to precisely edit the zebrafish genome to create single-nucleotide substitutions, which account for ~60% of human disease-causing mutations. Recently developed base editing nucleases provide an excellent alternate to CRISPR/Cas9-mediated homology dependent repair for generation of zebrafish with point mutations. A new set of cytosine base editors, termed BE4max and AncBE4max, demonstrated improved base editing efficiency in mammalian cells but have not been evaluated in zebrafish. Therefore, we undertook this study to evaluate their efficiency in converting C:G to T:A base pairs in zebrafish by somatic and germline analysis using highly active sgRNAs to twist and ntl genes. Our data demonstrated that these improved BE4max set of plasmids provide desired base substitutions at similar efficiency and without any indels compared to the previously reported BE3 and Target-AID plasmids in zebrafish. Our data also showed that AncBE4max produces fewer incorrect and bystander edits, suggesting that it can be further improved by codon optimization of its components for use in zebrafish.
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Programmable base editing in zebrafish using a modified CRISPR-Cas9 system.
The use of CRISPR/Cas9 to knockout genes in zebrafish has been well established. However, to better model many human diseases that are caused by point mutations, a robust methodology for generating desirable DNA base changes is still needed. Recently, Cas9-linked cytidine deaminases (base editors) evolved as a strategy to introduce single base mutations in model organisms. They have been used to convert cytidine to thymine at specific genomic loci. Here we describe a protocol for using the base editing system in zebrafish and its application to reproduce a single base mutation observed in human Ablepharon-Macrostomia Syndrome.
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Plant-Based Biosensors for Detecting CRISPR-Mediated Genome Engineering.
CRISPR/Cas has recently emerged as the most reliable system for genome engineering in various species. However, concerns about risks associated with the CRISPR/Cas technology are increasing on potential unintended DNA changes that might accidentally arise from CRISPR gene editing. Developing a system that can detect and report the presence of active CRISPR/Cas tools in biological systems is therefore very necessary. Here, we developed four real-time detection systems that can spontaneously indicate the presence of active CRISPR-Cas tools for genome editing and gene regulation including CRISPR/Cas9 nuclease, base editing, prime editing, and CRISPRa in plants. Using the fluorescence-based molecular biosensors, we demonstrated that the activities of CRISPR/Cas9 nuclease, base editing, prime editing, and CRISPRa can be effectively detected in transient expression via protoplast transformation and leaf infiltration (in Arabidopsis, poplar, and tobacco) and stable transformation in Arabidopsis.
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