Aug 2026· Tissue Engineering. Part C, Methods· pp.
19373384261477635
· 0 citations· 16 references
Medicine
Abstract
Efficient delivery of large gene-editing plasmids, such as the mCherry-CRISPR dCas9 system, into nucleus pulposus (NP) cells is a key step in generating sufficient cells for tissue-engineered intervertebral disc (IVD) constructs and other regenerative therapies for degenerative disc disease (DDD). However, the transfection and transduction of these environmentally sensitive cells remain challenging. This study aimed to identify the best protocol for delivery with minimal cytotoxicity and greatest efficiency. Transfection conditions in HEK293T cells were evaluated using Lipofectamine 3000, Lipofectamine Classic, and ViaFect at different reagent-to-DNA ratios and DNA amounts. Transfection efficiency was quantified by flow cytometry based on mCherry expression. Lentivirus was produced and concentrated by comparing PEG8000, a commercial Lenti-X concentrator, and ultracentrifugation. For NP cell transduction, polybrene and protamine sulfate were tested at multiple concentrations to maximize efficiency and viability. The optimized protocol was validated by delivering a CRISPR/dCas9 Synergistic Activation Mediator (SAM) system to activate endogenous FOXA2, and by seeding the resulting cells onto membranes to assess in vitro NP-like tissue formation. Lipofectamine 3000 at a 2:1 reagent-to-DNA ratio with 0.5 µg DNA per well yielded the highest transfection efficiency in HEK293T cells while minimizing cytotoxicity. Coprecipitation methods for lentiviral concentration, particularly the in-house PEG8000 concentrator, were better than ultracentrifugation. Protamine sulfate at 30 µg/mL yielded efficient NP cell transduction with higher viability than polybrene, as assessed by survival after antibiotic selection. Application of this protocol upregulated endogenous FOXA2 mRNA and protein expression, demonstrating functional efficacy. SAM-FOXA2 cells produced thicker tissue on membrane inserts than SAM controls, confirming compatibility of the protocol with downstream tissue engineering applications. Therefore, an optimized protocol balancing high transfection/transduction efficiency with minimized cytotoxicity was developed, supporting tissue-engineered IVD constructs and other CRISPR-based regenerative therapies for DDD.
It is concluded that bridging the gap between foundational CRISPR research and its real-world applications is imperative and future efforts should focus on democratizing tools via open-source platforms, advancing delivery systems, and fostering sustainable innovation through synthetic biology integration to fully realize the transformative potential of genome editing in organisms beyond model organisms.
S. Sarsaiya, Archana Jain, Jishuang Chen et al.· Biotechnology Advances· 2 citations
It is argued that formation of a tumour-intrinsic niche is a prerequisite for BRAF-mutant CRC seeding to distant organs and that interference with niche formation may help avoid metastatic relapse.
J. Bugter, L. El Bouazzaoui, E. Küçükköse et al.· bioRxiv· 2 citations
This review summarizes emerging therapeutic strategies for EOC, their mechanisms of action, and their potential to overcome treatment resistance, and covers molecularly targeted therapies, immunotherapies, metabolic and epigenetic approaches, cellular and gene therapies, targeted drug-delivery systems, and locoregional and physical modalities.
Zofia Pietrasik, Mikołaj Kapała, Joanna Pietrasik et al.· Cancers· 0 citations
Genetic engineering (GE) and gene editing may endow traits to trees such as increased biomass and the production of novel biomaterials. Long-lived organisms such as trees might be subject to biotechnology-related risks that could be different than those of annual row crops. Those risks could be relevant to production in engineered plantations and beyond plantations to natural forests. Therefore, appropriate risk regulation is important to assure biosafety of commercialized engineered trees. In addition to gene flow via sexual reproduction, vegetative reproduction might play an additional role in environmental "exposure" risk relative to transgene dispersal in GE tree plantations. While vegetative reproduction is beneficial for preserving desired genetic traits during tree propagation, it may lead to proximal clonal spread in the field. Although the environmental risks associated with vegetative reproduction of GE trees are recognized in commercial forestry, there are few field-based environmental risk assessment (ERA) studies on dispersal risks of self-propagated GE trees. GE or gene editing of target genes involved in the vegetative propagation processes may be useful to mitigate environmental risks of clonal spread through vegetative reproduction. This review provides updates for recent field test results of GE and gene edited trees. Gene candidates related to vegetative reproduction including adventitious shooting (AS) and adventitious rooting (AR) are discussed herein as a means to mitigate unintended clonal spread from GE tree plantations.
Findings establish Cas7-11 as a precise and efficient RNA knockdown tool for functional studies in embryonic development and stem cell biology, providing a versatile alternative to DNA-based gene-editing approaches.
Huan Yan, Imtiaz Ul Hassan, Kai Yan et al.· Cell & Bioscience· 0 citations
A new method for surgically removing training examples from a model reveals that as datasets grow, the link between what a model learns and what it produces dissolves.
MIT News · Artificial Intelligence· news.mit.eduAug 17, 2026