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User-Friendly Technology the Key to Gene-Editing's Bloom: Market for Gene-Editing Tools Estimated at $608m and Growing as New Applications Are Found.
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Gene Editing Provides Clues to Why Cancer Immunotherapy Often Fails.
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[Nanoblades: Pseudoviral shuttles for CRISPR-CAS9 delivery].
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Comparative analysis of lipid Nanoparticle-Mediated delivery of CRISPR-Cas9 RNP versus mRNA/sgRNA for gene editing in vitro and in vivo.
The discovery that the bacterial defense mechanism, CRISPR-Cas9, can be reprogrammed as a gene editing tool has revolutionized the field of gene editing. CRISPR-Cas9 can introduce a double-strand break at a specific targeted site within the genome. Subsequent intracellular repair mechanisms repair the double strand break that can either lead to gene knock-out (via the non-homologous end-joining pathway) or specific gene correction in the presence of a DNA template via homology-directed repair. With the latter, pathological mutations can be cut out and repaired. Advances are being made to utilize CRISPR-Cas9 in patients by incorporating its components into non-viral delivery vehicles that will protect them from premature degradation and deliver them to the targeted tissues. Herein, CRISPR-Cas9 can be delivered in the form of three different cargos: plasmid DNA, RNA or a ribonucleoprotein complex (RNP). We and others have recently shown that Cas9 RNP can be efficiently formulated in lipid-nanoparticles (LNP) leading to functional delivery in vitro. In this study, we compared LNP encapsulating the mRNA Cas9, sgRNA and HDR template against LNP containing Cas9-RNP and HDR template. Former showed smaller particle sizes, better protection against degrading enzymes and higher gene editing efficiencies on both reporter HEK293T cells and HEPA 1-6 cells in in vitro assays. Both formulations were additionally tested in female Ai9 mice on biodistribution and gene editing efficiency after systemic administration. LNP delivering mRNA Cas9 were retained mainly in the liver, with LNP delivering Cas9-RNPs additionally found in the spleen and lungs. Finally, gene editing in mice could only be concluded for LNP delivering mRNA Cas9 and sgRNA. These LNPs resulted in 60 % gene knock-out in hepatocytes. Delivery of mRNA Cas9 as cargo format was thereby concluded to surpass Cas9-RNP for application of CRISPR-Cas9 for gene editing in vitro and in vivo.
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Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells.
Long-range correction strategies require ex vivo activation of hematopoietic stem and progenitor cells (HSPCs) to engage the homology-directed repair (HDR) mechanism, but prolonged culture causes harmful cellular responses, reducing the long-term functionality of gene-edited (GE) HSPCs. Here, we present a protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human HSPCs. We describe steps for HSPC thawing, ex vivo treatments, gene editing, and downstream in vitro and in vivo analyses to assess the functionality of GE-HSPCs. For complete details on the use and execution of this protocol, please refer to della Volpe et al.1.
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The Brave New World of Gene Editing and Molecular Medicine.
Gene therapy has long been a promise of molecular biology. So far, that promise has largely been unrealized. The advent of gene editing using technology adapted from bacteria may finally usher in the era of gene therapy.
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Biomimetic Mineralized CRISPR/Cas RNA Nanoparticles for Efficient Tumor-Specific Multiplex Gene Editing.
CRISPR/Cas9 systems have great potential to achieve sophisticated gene therapy and cell engineering by editing multiple genomic loci. However, to achieve efficient multiplex gene editing, the delivery system needs adequate capacity to transfect all CRISPR/Cas9 RNA species at the required stoichiometry into the cytosol of each individual cell. Herein, inspired by biomineralization in nature, we develop an all-in-one biomimetic mineralized CRISPR/Cas9 RNA delivery system. This system allows for precise control over the coencapsulation ratio between Cas9 mRNA and multiple sgRNAs, while also exhibiting a high RNA loading capacity. In addition, it enhances the storage stability of RNA at 4 °C for up to one month, and the surface of the nanoparticles can be easily functionalized for precise targeting of RNA nanoparticles in vivo at nonliver sites. Based on the above characteristics, as a proof-of-concept, our system was able to achieve significant gene-editing at each target gene (Survivin: 31.9%, PLK1: 24.41%, HPV: 23.2%) and promote apoptosis of HeLa cells in the mouse model, inhibiting tumor growth without obvious off-target effects in liver tissue. This system addresses various challenges associated with multicomponent RNA delivery in vivo, providing an innovative strategy for the RNA-based CRISPR/Cas9 gene editing.
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Non-chromatographic purification of guide RNA for gene-editing experiments.
CRISPR-Cas12a gene editing technology is gaining momentum as a powerful tool for many biochemical and medicinal applications. The technology requires guide RNA, which is typically made using solid phase synthesis and purified by HPLC. The latter is often the most complex and time-consuming element of the synthetic process. This communication describes a non-chromatographic method for purification of synthetic RNAs. The method consists of five steps and yields target RNA in over 80% purity, which adheres to the FDA's standard for gene editing applications. The non-chromatographic RNA purification approach was applied to synthesize guide RNA targeting the GFP gene. Its purity was analyzed by analytical HPLC. Its functional fidelity was tested in CRISPR-Cas12a experiments in solution and live mammalian cells.
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Construction of a CRISPR/FnCas12a multi-sites editing system for inhibiting proliferation of Bombyx mori nuclearpolyhedrosisvirus.
In recent years, Cas12a, a new member of the CRISPR family, has been found to have both DNase and RNase activities, have a simple structure, and a single promoter can simultaneously initiate multiple crRNAs, making the CRISPR/Cas12a editing system more advantageous in terms of structure and mechanism of action. Our team has successfully constructed Cas12a system that can be used in silkworm. Cas12a can be used to edit the multiple target sites. In production, a lot of factors can affect the production of silk industry. In order to make the silkworm resistant to the virus, using gene editing technology to knock out key genes for replication and proliferation in the Bombyx mori nuclearpolyhedrosisvirus (BmNPV) genome. Multiple sites on the BmNPV genome were selected as the target sites. We constructed the multi-sites expression vector of gie1-M (361 bp, 597 bp, 927 bp of ie-1) that edited multiple sites of BmNPV ie-1. The effects of multi-sites editing system on the proliferation and replication of the virus after the BmNPV genome was knocked out were examined. The results show that compared with CRISPR/FnCas12a single-site editing (gie1), multi-sites editing (gie1-M) can knock out the BmNPV genome more effectively and have a higher inhibitory effect on virus replication and proliferation. This system can provide a new direction for the breeding of silkworm resistant materials, and it can also lay a good technical platform for the identification and research of biological gene function.
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CRISPR/Cas9 to Silence Long Non-Coding RNAs.
Knockout (KO) of long non-coding RNAs (lncRNAs) enables functional characterization of this still poorly described group of transcripts. One of the most efficient and simplest methods to achieve complete KO of a lncRNA is by employing CRISPR/Cas gene editing. As most lncRNAs are not well annotated, their individual functional regions are often not defined, and the majority of the transcripts are not affected by single nucleotide mutations. Therefore, CRISPR/Cas KO is more challenging for lncRNAs as compared to KO of protein coding genes. Strategies for lncRNAs KO include complete removal of the entire gene, removal of the promoter and transcriptional start site, abolishing exon-exon junctions, or removing the transcriptional termination site. Here, we describe the methodology to perform CRISPR/Cas9 KO of lncRNAs in vitro using electroporation as the method of transfection of presynthesized single guide RNAs (sgRNAs) and Cas9 enzyme.
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