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Expanding base editing scope to near-PAMless with engineered CRISPR/Cas9 variants in plants.
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SWISS: multiplexed orthogonal genome editing in plants with a Cas9 nickase and engineered CRISPR RNA scaffolds.
We describe here a CRISPR simultaneous and wide-editing induced by a single system (SWISS), in which RNA aptamers engineered in crRNA scaffold recruit their cognate binding proteins fused with cytidine deaminase and adenosine deaminase to Cas9 nickase target sites to generate multiplexed base editing. By using paired sgRNAs, SWISS can produce insertions/deletions in addition to base editing. Rice mutants are generated using the SWISS system with efficiencies of cytosine conversion of 25.5%, adenine conversion of 16.4%, indels of 52.7%, and simultaneous triple mutations of 7.3%. The SWISS system provides a powerful tool for multi-functional genome editing in plants.
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[Advancements in CRISPR-Cas9 for Fanconi anemia].
Fanconi anemia (FA) is a hereditary bone marrow failure syndrome that is characterized by genomic instability and heightened sensitivity to DNA cross-linking agents. In recent years, the CRISPR-Cas9 technology has exhibited groundbreaking progress in the field of gene therapy for FA. The traditional CRISPR-Cas9 technology has been successfully applied in FA gene editing. Further, single-base editing technology, based on the CRISPR/Cas9 system, performs precise and efficient gene repair for prevalent gene mutations in patients with FA. The prime editing technology provides new possibilities for gene editing; however, its application in FA has not been initiated. Despite significant advancements in FA gene editing technology, several challenges remain, including the collection of sufficient hematopoietic stem cells, the risk of increased tumorigenesis postgene editing, chromosomal instability, and off-target effects. Future research is recommended to focus on optimizing sgRNA and Cas9 nucleases, designing stricter PAM sequences to reduce off-target effects, and devising personalized gene editing strategies. Further, ethical and regulatory issues as well as long-term follow-ups are crucial priorities for future gene editing work. With continuous technological advancements and in-depth clinical trials, we expect more breakthroughs in FA treatment using the CRISPR-Cas9 technology in the future. This article reviews the latest research progress of CRISPR technology in FA treatment and analyzes the advantages and disadvantages of this technology in FA gene therapy.
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Rewriting DNA in the body lowers cholesterol.
Verve Therapeutics says its base-editing approach may help prevent heart disease in many people.
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Treating Cardiovascular Disease with Liver Genome Engineering.
PURPOSE OF REVIEW: This review examines recent progress in somatic genome editing for cardiovascular disease. We briefly highlight new gene editing approaches, delivery systems, and potential targets in the liver. RECENT FINDINGS: In recent years, new editing and delivery systems have been applied successfully in model organisms to modify genes within hepatocytes. Disruption of several genes has been shown to dramatically lower plasma cholesterol and triglyceride levels in mice as well as non-human primates. More precise modification of cardiovascular targets has also been achieved through homology-directed repair or base editing. Improved viral vectors and nanoparticle delivery systems are addressing important delivery challenges and helping to mitigate safety concerns. Liver-directed genome editing has the potential to cure both rare and common forms of cardiovascular disease. Exciting progress is already being made, including promising results from preclinical studies and the initiation of human gene therapy trials.
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BASELINE: a CRISPR base editing platform for mammalian-scale single-cell lineage tracing.
A cell's fate is shaped by its inherited state, or lineage, and the ever-shifting context of its environment. CRISPR-based recording technologies are a promising solution for mapping the lineage of a developing system; however, challenges remain regarding single-cell recovery, engineering complexity, and scale. Here, we introduce BASELINE, which uses base editing to generate high-resolution lineage trees in conjunction with single-cell profiling. BASELINE uses the Cas12a adenine base editor to irreversibly edit nucleotides across target arrays built from 50 synthetic target sites, which are integrated multiple times into a cell's genome. We demonstrate that BASELINE accumulates lineage-specific marks over a wide range of biologically relevant intervals, recording more than 4300 bits of information in a model of pancreatic cancer, a 50-fold increase over existing technologies. Single-cell sequencing reveals high-fidelity capture of these recorders, averaging 29 cell divisions captured per lineage, within the estimated range of mammalian development. We expect BASELINE to apply to a wide range of lineage-tracing projects in development and disease, especially those in which cellular engineering makes small, more distributed systems challenging.
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Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells.
Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.
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Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants.
Streptococcus pyogenes Cas9 (SpCas9) is the most widely used genome editing tool in plants. The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif (PAM), significantly limiting its scope of application. Recently, five SpCas9 variants, SpCas9-NRRH, SpCas9-NRCH, SpCas9-NRTH, SpG, and SpRY, were developed to recognize non-canonical PAMs in human cells. In this study, these variants were engineered for plant genome editing, and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice (Oryza sativa) by stable transformation. Moreover, both cytosine base editors using a rat APOBEC1 or a human APOBEC3a and adenine base editors using a directly evolved highly compatible TadA∗-8e deaminase were developed from these SpCas9 variants. Our results demonstrated that the developed SpCas9 variants-based base editors readily generated conversions between C∙G and T∙A in the target sites with non-canonical PAMs in transgenic rice lines. Collectively, the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants.
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CRISPR 2.0: Expanding the genome engineering Toolbox for epigenetics, RNA editing, and molecular diagnostics.
Non-canonical CRISPR systems adaptation has led to genome editing through nucleases, and the development of transcriptional and epigenetic regulation, transcriptome editing, and molecular diagnostics has resulted in a diversified set of tools-CRISPR 2.0. In this review, the author summarizes the mechanisms and recent engineering advances of (i) dCas9-based epigenetic effectors, (ii) RNA-targeting Cas13 systems and engineered RNA editors, (iii) DNA base editors and prime editors, and (iv) CRISPR-powered diagnostic platforms and their translational readiness. There is a critical comparison of the various approaches (e.g., RNAi/ASO versus Cas13-based methods; base editing versus prime editing) along with practical translational considerations such as delivery technologies, safety (off-target/edit windows, mosaicism), and regulatory pathways which are evaluated. Three concise case studies refer to map laboratory evidence to clinical or near-clinical outcomes and the ethical and governance discussion is widened to include global access, intellectual property and equity in deployment. Finally, the authors classify technologies according to their level of readiness - diagnostics and some ex-vivo therapeutic approaches are already in or very close to clinical use, chosen in-vivo editing methods are undergoing early trials, and AI-assisted nuclease design is still mostly theoretical but is getting better fast. This comprehensive viewpoint is intended to help researchers and physicians understand which CRISPR tools are most likely to be translated soon and where more validation is required.
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DNA base editing corrects common hemophilia A mutations and restores factor VIII expression in in vitro and ex vivo models.
BACKGROUND: Replacement and nonreplacement therapies effectively control bleeding in hemophilia A (HA) but imply lifelong interventions. Authorized gene addition therapy could provide a cure but still poses questions on durability. FVIIIgene correction would definitively restore factor (F)VIII production, as shown in animal models through nuclease-mediated homologous recombination (HR). However, low efficiency and potential off-target double-strand break still limit HR translatability. OBJECTIVES: To correct common model single point mutations leading to severe HA through the recently developed double-strand break/HR-independent base editing (BE) and prime editing (PE) approaches. METHODS: Screening for efficacy of BE/PE systems in HEK293T cells transiently expressing FVIII variants and validation at DNA (sequencing) and protein (enzyme-linked immunosorbent assay; activated partial thromboplastin time) level in stable clones. Evaluation of rescue in engineered blood outgrowth endothelial cells by lentiviral-mediated delivery of BE. RESULTS: Transient assays identified the best-performing BE/PE systems for each variant, with the highest rescue of FVIII expression (up to 25% of wild-type recombinant FVIII) for the p.R2166∗ and p.R2228Q mutations. In stable clones, we demonstrated that the mutation reversion on DNA (∼24%) was consistent with the rescue of FVIII secretion and activity of 20% to 30%. The lentiviral-mediated delivery of the selected BE systems was attempted in engineered blood outgrowth endothelial cells harboring the p.R2166∗ and p.R2228Q variants, which led to an appreciable and dose-dependent rescue of secreted functional FVIII. CONCLUSION: Overall data provide the first proof-of-concept for effective BE/PE-mediated correction of HA-causing mutations, which encourage studies in mouse models to develop a personalized cure for large cohorts of patients through a single intervention.
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