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Genome editing, especially via the simple and versatile type II CRISPR/Cas9 system, offers an effective avenue to precisely control cell fate, an important aspect of tissue regeneration. Unfortunately, most CRISPR/Cas9 non-viral delivery strategies only utilise micro-/nano-particle delivery methods. While these approaches provide reasonable genomic editing efficiencies, their systemic delivery may lead to undesirable off-target effects. For in vivo applications, a more localized and sustained delivery approach may be useful, particularly in the context of tissue regeneration. Here, we developed a scaffold that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and non-viral manner. Specifically, using mussel-inspired bioadhesive coating, polyDOPA-melanin (pDOPA), we absorbed Cas9:sgRNA lipofectamine complexes onto bio-mimicking fiber scaffolds. To evaluate the genome-editing efficiency of this platform, U2OS.EGFP cells were used as the model cell type. pDOPA coating was essential in allowing Cas9:sgRNA lipofectamine complexes to adhere onto the scaffolds with a higher loading efficiency, while laminin coating was necessary for maintaining cell viability and proliferation on the pDOPA-coated fibers for effective gene editing (21.5% editing efficiency, p < 0.001). Importantly, U2OS.EGFP cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds via reverse transfection. Overall, we demonstrate the efficacy of such fiber scaffolds in providing localized, sustained and non-viral delivery of Cas9:sgRNA complexes. Such genome editing scaffolds may find useful applications in tissue regeneration. Statement of Significance Currently, there is a lack of effective non-viral means to deliver CRISPR/Cas9 components for genome editing. Most existing approaches only utilize micro-/nano-particles by injection or systemic delivery, which may lead to undesirable off-target effects. Here, we report a platform that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and sustained manner. We used mussel-inspired bioadhesive coating to functionalize the bio-mimicking fiber scaffolds with Cas9:sgRNA lipofectamine complexes, to allow effective gene editing for the cells seeded on the scaffolds. Importantly, the cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds. Such genome editing scaffolds may find useful applications in tissue regeneration.
This article was published in the following journal.
Name: Acta biomaterialia
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Protein components of the CRISPR-CAS SYSTEMS for anti-viral defense in ARCHAEA and BACTERIA. These are proteins that carry out a variety of functions during the creation and expansion of the CRISPR ARRAYS, the capture of new CRISPR SPACERS, biogenesis of SMALL INTERFERING RNA (CRISPR or crRNAs), and the targeting and silencing of invading viruses and plasmids. They include DNA HELICASES; RNA-BINDING PROTEINS; ENDONUCLEASES; and RNA and DNA POLYMERASES.
Adaptive antiviral defense mechanisms, in archaea and bacteria, based on DNA repeat arrays called CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR elements) that function in conjunction with CRISPR-ASSOCIATED PROTEINS (Cas proteins). Several types have been distinguished, including Type I, Type II, and Type III, based on signature motifs of CRISPR-ASSOCIATED PROTEINS.
Semi-synthetic complex derived from nucleic-acid free viral particles. They are essentially reconstituted viral coats, where the infectious nucleocapsid is replaced by a compound of choice. Virosomes retain their fusogenic activity and thus deliver the incorporated compound (antigens, drugs, genes) inside the target cell. They can be used for vaccines (VACCINES, VIROSOME), drug delivery, or gene transfer.
A catalytic subunit of the NuA4 histone acetyltransferase complex that functions in transcriptional activation of genes by acetylation of nucleosomal HISTONES H4 and H2A, altering nucleosome-DNA interactions and interaction of the modified histones with other activating transcription factors. It may control gene expression changes associated with oncogene and proto-oncogene mediated growth induction, tumor suppressor mediated growth arrest; CELL AGING; APOPTOSIS; and DNA REPAIR. It is polyubiquitinated and degraded during HIV-1 infection through its interaction with the viral TAT PROTEIN.
Homer proteins belong to a family of adaptor and scaffold proteins which include Homer1, Homer2 and Homer3. Homer1 and Homer2 play a role in the regulation of calcium homeostasis, whereas Homer3 functions in stimulating changes in actin dynamics in neurons and T-cells. Homer proteins are best known as scaffold proteins at the post-synaptic density where they facilitate synaptic signaling. They function as a molecular switch in metabotropic glutamate receptor (MGluR) signaling, and are associated with human Fragile X syndrome.
Bioinformatics is the application of computer software and hardware to the management of biological data to create useful information. Computers are used to gather, store, analyze and integrate biological and genetic information which can then be applied...