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Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.
Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx4cv mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.
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LRRCA8A and ANO1 contribute to serum-induced VRAC in a Ca2+-dependent manners.
The volume-regulated anion channel (VRAC) plays a central role in maintaining cell volume in response to osmotic stress. Leucine-rich repeat-containing 8A (LRRC8A) was recently identified as an essential component of VRAC although other Cl- channels were also suggested to contribute to VRAC. VRAC is activated when a cell is challenged with a hypotonic environment or even in isotonic conditions challenged with different stimuli. It is not clear how VRAC is activated and whether activation of VRAC in hypotonic and isotonic conditions share the same mechanism. In this present study, we investigated relative contribution of LRRC8A and anoctamin 1(ANO1) to VRAC currents activated by fetal bovine serum (FBS) in isotonic condition, and studied the role of intracellular Ca2+ in this activation. We used CRISPR/Cas9 gene editing approach, electrophysiology, and pharmacology approaches to show that VRAC currents induced by FBS is mostly mediated by LRRC8A in HEK293 cells, but also with significant contribution from ANO1. FBS induces Ca2+ transients and these Ca2+ signals are required for the activation of VRAC by serum. These findings will help to further understand the mechanism in activation of VRAC.
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Feasible development of stable HEK293 clones by CRISPR/Cas9-mediated site-specific integration for biopharmaceuticals production.
OBJECTIVE: To inspect the feasibility of recombinant stable HEK293 cell lines development for biopharmaceuticals production using CRISPR/Cas9-mediated site-specific integration. RESULTS: Using EGFP as a model protein, we first confirmed that the 'safe harbor' AAVS1 locus could be successfully targeted and the exogenous genes could be integrated through homology-directed repair induced by CRISPR/Cas9 technology. Then we constructed a donor plasmid harboring CTLA4Ig gene with an upstream CMV promoter and a downstream puromycin N-acetyltransferase gene to accelerate the efficient integration and selection of CTLA4Ig expression clones. After puromycin enrichment, the transfected pool was diluted for single clone selection, and 12 recombinant clones with CTLA4Ig expression were finally selected with a targeting efficiency of 25.8%. Productivity assay demonstrated that a frequency of 83.3% of selected clone were of consistent productivities, thus illustrating the high efficiency and success rate of this strategy. CONCLUSIONS: CRISPR/Cas9 mediated site-specific integration is an efficient and reliable tool to establishment recombinant stable HEK293 cell lines for both academic and industrial applications.
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The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit.
CRISPR-Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR-Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR-Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR-Cas systems. This diversity has enabled further expansion of CRISPR-Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR-Cas systems.
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CRISPR-edited DPSCs constitutively expressing BDNF enhance dentin regeneration in injured teeth.
Dental caries, a prevalent global health issue, results from complex bacterial interactions. In response to harmful stimuli, a desirable outcome for the tooth is the formation of tertiary dentin, a protective reparative process that generates new hard tissue. This reparative dentinogenesis is associated with significant inflammation, which triggers the recruitment and differentiation of dental pulp stem cells (DPSCs). Previously, we have demonstrated that brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin receptor kinase B (TrkB), key mediators of neural functions, are activated during the DPSC-mediated dentin regeneration process. In this study, we further define the role of inflammation in this process and apply stem cell engineering to enhance dentin regeneration in injured teeth. Our data show that TrkB expression and activation in DPSCs rapidly increase during odontogenic differentiation, further amplified by inflammatory inducers and mediators such as tumor necrosis factor alpha (TNFα), lymphotoxin-alpha, and lipopolysaccharide. An in vivo dentin formation assessment was conducted using a mouse pulp-capping/caries model, where Clustered Regularly Interspaced Short Palindromic Repeats-engineered DPSCs overexpressing BDNF were transplanted into inflamed pulp tissue. This transplantation significantly enhanced dentin regeneration in injured teeth. To further explore potential downstream pathways, we conducted transcriptomic profiling of TNFα-treated DPSCs, both with and without TrkB antagonist cyclotraxin-B. The results revealed significant changes in gene expression related to immune response, cytokine signaling, and extracellular matrix interactions. Taken together, our study advances our understanding of the role of BDNF in dental tissue engineering using DPSCs and identifies potential therapeutic avenues for improving dental tissue repair and regeneration strategies.
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Opportunities in biotechnology.
Strategies for biotechnology must take account of opportunities for research, innovation and business growth. At a regional level, public-private collaborations provide potential for such growth and the creation of centres of excellence. By considering recent progress in areas such as genomics, healthcare diagnostics, synthetic biology, gene editing and bio-digital technologies, opportunities for smart, strategic and specialised investment are discussed. These opportunities often involve convergent or disruptive technologies, combining for example elements of pharma-science, molecular biology, bioinformatics and novel device development to enhance biotechnology and the life sciences. Analytical applications use novel devices in mobile health, predictive diagnostics and stratified medicine. Synthetic biology provides opportunities for new product development and increased efficiency for existing processes. Successful centres of excellence should promote public-private business partnerships, clustering and global collaborations based on excellence, smart strategies and innovation if they are to remain sustainable in the longer term.
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From iPSC towards cardiac tissue-a road under construction.
The possibility to generate induced pluripotent stem cells (iPSC) opens the way to generate virtually all cell types of our human body. In combination with modern gene editing techniques like CRISPR/CAS, a new set of powerful tools becomes available for life science. Scientific fields like genotype and cell type-specific pharmacology, disease modeling, stem cell biology, and developmental biology have been dramatically fostered and their faces have been changed. However, as golden as the age of iPSC-derived cells and their manipulation has started, the shine begins to tarnish. Researchers face more and more practical problems intrinsic to the system. These problems are related to the specific culturing conditions which are not yet sufficient to mimic the natural environment of native stem cells differentiating towards adult cells. However, researchers work hard to uncover these factors. Here, we review a common standard approach to generate iPSCs and transduce these to iPSC cardiomyocytes. Further, we review recent achievements and discuss their current limitations and future perspectives. We are on track, but the road is still under construction.
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Miniaturized scalable arrayed CRISPR screening in primary cells enables discovery at the single donor resolution.
High-efficiency gene editing in primary human cells is critical for advancing therapeutic development and functional genomics, yet conventional electroporation platforms often require high cell input and are poorly suited to parallelized experiments. Here we introduce a next-generation digital microfluidics (DMF) electroporation platform that enables high-throughput, low-input genome engineering using discrete droplets manipulated on a planar electrode array. The system supports 48 independently programmable reaction sites and integrates seamlessly with laboratory automation, allowing efficient delivery of CRISPR-Cas9 RNPs and mRNA cargo into as few as 3,000 primary human cells per condition. The platform was validated across diverse primary human cell types and cargo modalities, demonstrating efficient delivery of various cargo, with high rates of transfection, gene knockout via non-homologous end joining, and precise knock-in through homology-directed repair. To showcase its utility in functional genomics, we applied the platform to an arrayed CRISPR-Cas9 screen in chronically stimulated human CD4⁺ T cells, identifying novel regulators of exhaustion, including epigenetic and transcriptional modulators. These findings establish our DMF-based electroporation platform as a powerful tool for miniaturized genome engineering in rare or precious cell populations and provide a scalable framework for high-content genetic screening in primary human cells.
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CRISPR/Cas9 editing of the genome for cancer modeling.
The CRISPR/Cas9 revolution has democratized access to genome editing in many biological fields, including cancer research. Cancer results from the multistep accumulation of mutations that confer to the transformed cells certain biological hallmarks typical of the malignant phenotype. One of the major goals in cancer research is to characterize such mutations and assess their implication in the oncogenic process. Through CRISPR/Cas9 technology, genetic aberrations identified in a patient's tumor can now be easily recreated in experimental models, which can then be used for basic research or for more translational applications. Here we review the different CRISPR/Cas9 strategies that have been implemented to recapitulate oncogenic mutations in both in vitro and in vivo systems, including novel strategies to model tumor evolution and genetic heterogeneity.
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Generation of Mouse Model (KI and CKO) via Easi-CRISPR.
Recent development of Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) that utilizes long single-stranded DNA (lssDNA) of 0.2-2 kbases in length as donor templates to insert large segments of novel DNA sequences or to replace endogenous genes at precise locations in the genome has enabled CRISPR-assisted genome editing to make strides toward a more simple and rapid workflow. By leveraging the notion that short single-stranded DNA oligo (<200 bases) serves as efficient donor in mouse zygotes for facilitating HDR-mediated genome editing, Easi-CRISPR expands to use lssDNA as the donor which accelerates the timeline to as little as 2 months for creating most types of genetically engineered mouse models (F0). Our lab (CGERC) has adopted Easi-CRISPR for multiple loci to generate mouse models over the past three plus years since its introduction. Here, we use two genes as examples to illustrate a step-by-step protocol for generating two commonly used models, including a knock-in (insertion of a reporter gene plus GOI) as well as a conditional knock-out model (via exon floxing). This protocol will focus more on molecular biology aspect, particularly we demonstrate two recently developed methods for lssDNA procuration: (1) PCR-based Takara Bio kit with modifications; (2) plasmid-retrieval-based CRISPR-CLIP (CRISPR-Clipped LssDNA via Incising Plasmid). Both methods are devised to retain sequence fidelity in lssDNA generated. In addition, CRISPR-CLIP directly retrieves lssDNA from DNA plasmid without using restriction enzymes through a PCR-free system hence carries virtually no restriction on sequence complexity, further mitigating limitations discussed in the original Easi-CRISPR protocol. We have alternated the use between both methods when suitable and successfully generated lssDNA templates via CRISPR-CLIP up to 3.5 kbases patched with multiple highly repetitive sequences, which is otherwise challenging to maneuver. Along with certain other modified workflow presented herein, Easi-CRISPR can be adapted to be more straightforward while applicable to generate mouse models in broader scope. (Certain figures and text passages presented in this chapter are reproduced from Shola et al. (The CRISPR J 3(2):109-122, 2020), published by Mary Ann Libert, Inc).
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