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Inhibition of ADAMTS1 Expression by Lentiviral CRISPR/Cas9 Gene Editing Technology.
The continuous improvement of gene editing tools has allowed a major revolution in biological sciences. Although a variety of gain and loss-of-function approaches have been widely used for the last decades, some limitations arose from non-specific targeting or lack of complete inhibition of the gene of interest. CRISPR/Cas9 editing technology introduced new and significant advantages because it can directly modify the gene of interest and completely blocks its expression.In the context of cancer studies, the heterogeneity of the tumor microenvironment requires comprehensive approaches to unveil the contribution of multiple genes. For example, a deeper understanding of the biology of proteases such as ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motifs) will improve our perspective of complex phenomena affected by extracellular matrix remodeling, including embryonic development, angiogenesis, immune infiltration, metastasis, and tumor plasticity. Here, we present a method using CRISPR/Cas9 technology to inhibit the expression of the representative ADAMTS1 in cancer cells. Following the first steps of gene edition, we pursue further selection of silenced cells and provide a detailed description of sequence analysis and validation assays. This method leads to inactivation of ADAMTS1 in cancer cells, providing a relevant biological tool that will allow subsequent in vivo and in vitro ADAMTS1 functional analysis.
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Prime editing sensors enable multiplexed genome editing.
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Design of function-regulating RNA via deep learning and AlphaFold 3.
RNAs are programmable macromolecules that play diverse regulatory roles in living organisms. However, the intricate structure-function relationships underlying their regulatory activities pose significant challenges for RNA design. Here, we introduce a computational framework that integrates deep learning and energy-based methods to enhance the sequence diversity of sgRNAs designs. Our approach demonstrates high editing efficiencies of up to 75% for gene knockouts, 100% for large fragment deletions, and 62.5% for multiplex gene editing using the designed sgRNAs. Molecular dynamic simulations suggested the stability of DNA-RNA-protein complex is essential to the functionality of designed RNAs. Moreover, we reveal that the confidence metrics of AlphaFold 3 can effectively distinguish functional sequences, enabling one-shot design of crRNAs. This work presents an efficient strategy for designing regulatory RNAs with complex interactions and establishes the potential of AlphaFold 3 in advancing RNA design.
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[New-generation editing].
Recent work on bacterial insertion sequences reveals that some of them use an RNA sequence (called Bridge RNA or Seek RNA) to define both donor and target DNA specificity. This opens the way to easy insertion of kilobase DNA sequences at pre-defined sites in the genome, announcing a host of new possibilities. The system still needs a lot of tweaking, as it has only been demonstrated in bacteria, but it holds great promise for genome editing and engineering.
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Lipid nanoparticle formulation for gene editing and RNA-based therapies for glioblastoma.
BACKGROUND: Glioblastoma (GBM), one of the deadliest cancers, resists current therapies, with drug development hindered by its high heterogeneity. However, GBM consistently relies on microRNA-10b (miR-10b), a key driver of glioma growth and a promising therapeutic target. miR-10b gene editing represents a potential treatment, but effective delivery strategies for gene editing systems in GBM remain unexplored. METHODS: We developed lipid nanoparticles (LNPs) encapsulating Cas9 mRNA and a miR-10b-targeting sgRNA (termed miRTEN). miRTEN was tested in glioma stem cells (GSCs) and orthotopic GBM models to assess therapeutic efficacy, immune responses, and safety. RESULTS: Intracerebroventricular injections of miRTEN enabled broad and durable Cas9 mRNA expression and miR-10b gene editing in tumor core and invasive areas across diverse GBM models. miRTEN significantly suppressed tumor growth, reduced GSC proliferation and viability, with therapeutic outcomes correlating with dose-dependent miR-10b suppression. Combining miRTEN with temozolomide (TMZ) further enhanced tumor suppression, overcoming TMZ resistance and improving survival. In immunocompetent models, miRTEN activated antitumor immune responses, increased cytotoxic CD8+ T cells infiltration, and promoted durable immune memory, enabling tumor rejection upon rechallenge. Safety assessments demonstrated that miRTEN selectively targets GBM cells, sparing normal brain tissues and causing no significant off-target toxicity. CONCLUSION: As in vivo CRISPR-based drugs advance toward clinical applications, our findings demonstrate the potential of LNPs-mediated CRISPR-Cas9 systems for targeted miR-10b editing and, more generally, gene editing and RNA therapies for GBM. miRTEN monotherapy, as well as its combination with standard care, offers a promising, safe, and effective approach to improving outcomes in GBM.
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Developing a UV-visible reporter-assisted CRISPR/Cas9 gene editing system to alter flowering time in Chrysanthemum indicum.
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Protocol for HiBiT tagging endogenous proteins using CRISPR-Cas9 gene editing.
We present a method of in vitro/in vivo protein detection by pairing CRISPR-Cas9 genome editing with the NanoBiT system. We describe steps for cell culturing, in vitro CRISPR-Cas9 ribonucleoprotein delivery, cell monitoring, efficiency assessments, and edit analysis through HiBiT assays. We then detail procedures to determine edit specificity through genomic DNA analysis, small interfering RNA reverse transfection, and HiBiT blotting. This protocol is simple to execute and multifunctional, and it enables high-throughput screens on endogenous proteins to be conducted with ease.
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Comparative analysis of single and dual sgRNA CRISPR/Cas9 ribonucleoprotein strategies for efficient Park7 gene editing in primary mouse embryonic fibroblasts.
BACKGROUND: Parkinson's disease (PD) is a progressive neurological condition that predominantly affects dopaminergic neurons in the substantia nigra. Mutations or deletions in the Park7 gene may cause a rare autosomal recessive form of early-onset PD. Generating reliable cellular models with specific Park7 disruption is crucial for elucidating the molecular mechanisms underlying PD pathogenesis. METHODS AND RESULTS: This study developed a plasmid-free CRISPR/Cas9 genome-editing method for targeting the Park7 gene in primary mouse embryonic fibroblasts (MEFs) using single- and dual-sgRNA-mediated ribonucleoprotein (RNP) system. Gene modification through the single sgRNA strategy was confirmed by the detection of indels by T7 endonuclease assay, whereas gene editing by the dual sgRNA strategy was validated through detection of large deletions by junctional PCR and Sanger sequencing. Deep amplicon sequencing showed an overall editing frequency of 50.68% and 24.87% for single-sgRNA and dual-sgRNA editing strategies, respectively. While single-sgRNA editing predominantly generated small insertions and deletions, large deletions constituted the predominant editing outcome in the dual-sgRNA group, accounting for 22.23% of the total reads. Further, Western blot analysis demonstrated a 12.03 ± 7.6% reduction in PARK7 protein expression following single sgRNA transfection, whereas dual sgRNA transfection showed a significant reduction of 54.89 ± 9.1% (p < 0.0001). CONCLUSION: This study demonstrates a plasmid-free CRISPR/Cas9 RNP-based approach for targeted Park7 gene editing in primary MEFs. The results underline the importance of considering both the outcomes of genomic editing and the expression of functional protein when evaluating CRISPR/Cas9 approaches. This established approach sets the basis for the development of Park7-edited cellular models to study molecular mechanisms associated with PD and the subsequent development of disease-relevant models.
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Adapting and Testing ReMOT Control for Expanded CRISPR-Era Genome Functions in Non-model Insects.
The movement of the Drosophila yolk protein (DmYP) across the mosquito oocyte membrane was both fortuitous and puzzling; the cells that become future offspring--oocytes--are closed off to molecules that are not specifically recognized by a receptor, but there is no obvious ortholog of the yolk protein/receptor for DmYP in mosquitoes. Nonetheless, a small fragment of DmYP was sufficient to move the massive ribonucleoprotein complex of Cas9 and a guide RNA from the open circulatory system of a female mosquito into the mosquito oocyte for targeting of the germline DNA and heritable mutation. This procedure, known as ReMOT Control, is a robust method for CRISPR/Cas9-mediated gene knockdown that has been adapted for many orders of insects, for ticks, and even for several species of crustacean by first identifying a suitable peptide for oocyte uptake, then expressing Cas9 as a fusion protein with the peptide and finally performing adult injections with expressed, purified protein and guide RNA against a gene with a visible marker phenotype. In order to support the adaptation of this procedure widely among entomologists, herein, we provide the protocols to: (a) Identify a suitable peptide for any insect by identifying the receptor-binding region of vitellogenin. (b) Clone a nucleotide coding the peptide and a fluorescent protein into the commercially available Addgene plasmid pET28a/Cas9-cys to generate a fusion-protein encoding gene. (c) Express a fusion protein for specific delivery of ReMOT Cas9 to the ovaries of an insect of interest. (d) Adapt a generalized procedure for adult injection of insects targeting the hemolymph and detecting ovary translocation and heritable gene editing.
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EasyCloneYALI: Toolbox for CRISPR-Mediated Integrations and Deletions in Yarrowia lipolytica.
In order to unlock the full potential of Yarrowia lipolytica, as model organism and production host, simple and reliable tools for genome engineering are essential. In this chapter, the practical details of working with the EasyCloneYALI Toolbox are described.Highlights of the EasyCloneYALI Toolbox are high genome editing efficiencies, multiplexed Cas9-mediated knockouts, targeted genomic integrations into characterized intergenic loci, as well as streamlined and convenient cloning for both marker-based and marker-free integrative expression vectors.
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