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Targeted demethylation of cathepsin D via epigenome editing rescues pathology in Alzheimer's disease mouse model.
Background: Cathepsin D (Ctsd) has emerged as a promising therapeutic target for Alzheimer's disease (AD) due to its role in degrading intracellular amyloid beta (Aβ). Enhancing Ctsd activity could reduce Aβ42 accumulation and restore the Aβ42/40 ratio, offering a potential AD treatment strategy. Methods: This study explored Ctsd demethylation in AD mouse models using dCas9-Tet1-mediated epigenome editing. We identified dCas9-Tet1 as an effective tool for demethylating the endogenous Ctsd gene in primary neurons and in vivo brains. Results: Treatment with Ctsd-targeted dCas9-Tet1 in primary neurons overexpressing mutant APP (mutAPP) reduced Aβ peptide levels and the Aβ42/40 ratio. Additionally, in vivo demethylation of Ctsd via dCas9-Tet1 in 5xFAD mice significantly altered Aβ levels and alleviated cognitive and behavioral deficits. Conclusion: These findings offer valuable insights into developing epigenome editing-based gene therapy strategies for AD.
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The use of new CRISPR tools in cardiovascular research and medicine.
Many novel CRISPR-based genome-editing tools, with a wide variety of applications, have been developed in the past few years. The original CRISPR-Cas9 system was developed as a tool to alter genomic sequences in living organisms in a simple way. However, the functions of new CRISPR tools are not limited to conventional genome editing mediated by non-homologous end-joining or homology-directed repair but expand into gene-expression control, epigenome editing, single-nucleotide editing, RNA editing and live-cell imaging. Furthermore, genetic perturbation screening by multiplexing guide RNAs is gaining popularity as a method to identify causative genes and pathways in an unbiased manner. New CRISPR tools can also be applied to ex vivo or in vivo therapeutic genome editing for the treatment of conditions such as hyperlipidaemia. In this Review, we first provide an overview of the diverse new CRISPR tools that have been developed to date. Second, we summarize how these new CRISPR tools are being used to study biological processes and disease mechanisms in cardiovascular research and medicine. Finally, we discuss the prospect of therapeutic genome editing by CRISPR tools to cure genetic cardiovascular diseases.
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ACtivE: Assembly and CRISPR-Targeted in Vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time.
Thanks to its sophistication, the CRISPR/Cas system has been a widely used yeast genome editing method. However, CRISPR methods generally rely on preassembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose an alternative method, Assembly and CRISPR-targeted in vivo Editing (ACtivE), that only relies on in vivo assembly of linear DNA fragments for plasmid and donor DNA construction. Thus, depending on the user's need, these parts can be easily selected and combined from a repository, serving as a toolkit for rapid genome editing without any expensive reagent. The toolkit contains verified linear DNA fragments, which are easy to store, share, and transport at room temperature, drastically reducing expensive shipping costs and assembly time. After optimizing this technique, eight loci proximal to autonomously replicating sequences (ARS) in the yeast genome were also characterized in terms of integration and gene expression efficiencies and the impacts of the disruptions of these regions on cell fitness. The flexibility and multiplexing capacity of the ACtivE were shown by constructing a β-carotene pathway. In only a few days, >80% integration efficiency for single gene integration and >50% integration efficiency for triplex integration were achieved on Saccharomyces cerevisiae BY4741 from scratch without using in vitro DNA assembly methods, restriction enzymes, or extra cloning steps. This study presents a standardizable method to be readily employed to accelerate yeast genome engineering and provides well-defined genomic location alternatives for yeast synthetic biology and metabolic engineering purposes.
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Precision T cell correction platform for inborn errors of immunity.
CRISPR-Cas9 gene editing is a promising tool to correct pathogenic variants for autologous cell therapies targeting inborn errors of immunity (IEI). Current strategies, such as gene knockout or cDNA knockin, address many single-gene defects but can disrupt gene expression, highlighting the need for precise correction platforms. While transplanting corrected autologous hematopoietic stem cells is a curative approach, it is unsuitable for patients with advanced disease, inflammation, or acute infections. As correcting T cells is an alternative therapeutic strategy for lymphoid IEIs, we present an efficient T cell single-nucleotide variant (SNV) correction platform based on homology-directed repair (HDR). By using STAT1 gain-of-function, cartilage hair hypoplasia, deficiency of ADA2, and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy as IEI models, we demonstrate that our platform achieves up to 80% correction, with resultant functional correction of the disease phenotype in the selected models. Furthermore, we performed safety profiling using GUIDE-seq, single-cell RNA sequencing, long-read genome sequencing, and proteomics analysis and detected no genomic, transcriptomic, or proteomic aberrations. This study establishes HDR-based SNV editing as a portable method for developing clinical autologous T cell therapies and represents a promising step toward a broad-spectrum gene correction platform for treating diverse monogenic immune disorders.
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Precision editing to improve fruit traits: CRISPR/Cas into the picture.
Crop growth, quality, and yield can be adversely affected by various biotic and abiotic stresses. Crop characteristics can be improved with conventional breeding and other variation-based breeding strategies. However, these strategies are time as well as resource consuming and to overcome this, novel approaches are necessary. CRISPR/Cas technique allows to improve desired traits more efficiently and accurately by targeting specific genes. Genome editing has become more versatile with CRISPR/Cas systems and is a valuable tool to protect food security by developing commercial crops optimized for yield and nutritional quality. Researchers are able to target and edit stress response pathway genes to develop crops with increased tolerance to stress. A lack of regeneration protocols and sufficient genome sequencing data has restricted fruit editing to only a few fruits (tomatoes, citrus, apple, kiwi, banana, grapes, strawberries, watermelon, etc.). This review is focused on CRISPR/Cas applications on the nutritional aspects of fruit engineering along with the challenges and opportunities. Another aspect which will be covered is the use of CRISPR/Cas technology to improve fruit resilience to biotic and abiotic stress, but not at the cost of yield. We discuss the pros and cons of using this technology, such as unintended effects on fruit traits or public concerns about GMOs. We conclude that the application of CRISPR/Cas9 technology has the potential to be of great benefit to the agricultural industry not only to improve nutritional aspects but also to help reduce crop losses.
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Engineering and comparison of cas12a-based genome editing systems in plants.
While Cas9 and Cas12a are both RNA-guided endonucleases used for genome editing, only Cas12a is able to process pre-crRNA via its additional ribonuclease activity. This feature reduces the complexity of Cas12a versus Cas9-based genome editing systems thus providing an attractive alternative for generating site-specific mutations in plants. Here we aimed to improve the efficiency of the cas12a-based generation of two double-strand breaks flanking the open reading frame of a target gene, leading to its full deletion. To this end, we compared the relative impact of different components on cas12a-based gene deletion efficiency in three different eudicotyledons, Arabidopsis thaliana, Lotus japonicus, and Nicotiana benthamiana. We detected the highest cas12a-based editing efficiency with a combination of suitable promoters for crRNA and cas12a expression, a tandem terminator to control cas12a expression, a re-coded cas12a, adapted to the codon usage of Arabidopsis and engineered to carry introns, and encoding a Cas12a flanked by a nuclear localization signal at both ends. Our work revealed the high potential for improving cas12a-based genome editing systems for plant genetic research.
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CGMP Compliant Microfluidic Transfection of Induced Pluripotent Stem Cells for CRISPR-Mediated Genome Editing.
Inherited retinal degeneration is a term used to describe heritable disorders that result from the death of light sensing photoreceptor cells. Although we and others believe that it will be possible to use gene therapy to halt disease progression early in its course, photoreceptor cell replacement will likely be required for patients who have already lost their sight. While advances in autologous photoreceptor cell manufacturing have been encouraging, development of technologies capable of efficiently delivering genome editing reagents to stem cells using current good manufacturing practices (cGMP) are needed. Gene editing reagents were delivered to induced pluripotent stem cells (iPSCs) using a Zephyr microfluidic transfection platform (CellFE). CRISPR-mediated cutting was quantified using an endonuclease assay. CRISPR correction was confirmed via digital PCR and Sanger sequencing. The resulting corrected cells were also karyotyped and differentiated into retinal organoids. We describe use of a novel microfluidic transfection platform to correct, via CRISPR-mediated homology-dependent repair (HDR), a disease-causing NR2E3 mutation in patient-derived iPSCs using cGMP compatible reagents and approaches. We show that the resulting cell lines have a corrected genotype, exhibit no off-target cutting, retain pluripotency and a normal karyotype and can be differentiated into retinal tissue suitable for transplantation. The ability to codeliver CRISPR/Cas9 and HDR templates to patient-derived iPSCs without using proprietary transfection reagents will streamline manufacturing protocols, increase the safety of resulting cell therapies, and greatly reduce the regulatory burden of clinical trials.
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Take Risks and Constantly Challenge the Status Quo.
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Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells.
Genome editing often takes the form of either error-prone sequence disruption by non-homologous end joining (NHEJ) or sequence replacement by homology-directed repair (HDR). Although NHEJ is generally effective, HDR is often difficult in primary cells. Here, we use a combination of immunophenotyping, next-generation sequencing, and single-cell RNA sequencing to investigate and reprogram genome editing outcomes in subpopulations of adult hematopoietic stem and progenitor cells. We find that although quiescent stem-enriched cells mostly use NHEJ, non-quiescent cells with the same immunophenotype use both NHEJ and HDR. Inducing quiescence before editing results in a loss of HDR in all cell subtypes. We develop a strategy of controlled cycling and quiescence that yields a 6-fold increase in the HDR/NHEJ ratio in quiescent stem cells ex vivo and in vivo. Our results highlight the tension between editing and cellular physiology and suggest strategies to manipulate quiescent cells for research and therapeutic genome editing.
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Standard screening methods underreport AAV-mediated transduction and gene editing.
Conventional methods to discern adeno-associated virus (AAV) vector transduction patterns are based on high, stable expression of a reporter gene. As a consequence, conventionally described tropisms omit cell types that undergo transient transduction, or have low but undetectable levels of reporter expression. This creates a blind spot for AAV-based genome editing applications because only minimal transgene expression is required for activity. Here, we use editing-reporter mice to fill this void. Our approach sensitively captures both high and low transgene expression from AAV vectors. Using AAV8 and other serotypes, we demonstrate the superiority of the approach in a side-by-side comparison with traditional methods, demonstrate numerous, previously unknown sites of AAV targeting, and better predict the gene editing footprint after AAV-CRISPR delivery. We anticipate that this system, which captures the full spectrum of transduction patterns from AAV vectors in vivo, will be foundational to current and emerging AAV technologies.
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