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Agrobacterium-Mediated Transformation for Commercial Wheat Varieties.
Wheat is a crucial crop for global food security, and new breeding techniques face limitations due to low regeneration rates and a lack of transformable genotypes. We follow and adapt a robust Agrobacterium tumefaciens-mediated transformation system in spring commercial wheat varieties from Chile and Argentina grown in speed breeding conditions. By incorporating the developmental gene fusion GRF4-GIF1, we achieved successful regeneration of plantlets in different wheat varieties through CRISPR/Cas9-based gene editing. This advancement reduces genotype dependency, allowing broader use of genome-editing tools in commercial wheat varieties. In addition to delving into technical complexities, this contribution aims to advance fundamental understanding and practical applications in wheat genetics, serving as a valuable resource for researchers expanding their expertise.
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Transcriptional regulation by CRISPR/dCas9 in common wheat.
The application of CRISPR/Cas9 system for gene editing, as a technical coup for biotechnology, is worldwide and encompasses multiple of species. The inactivation of catalytical site in Cas9 (dCas9) has been reprogrammed as an effective approach to regulate the transcriptional level of target genes, especially for the functionally essential genes and redundant genes. Here, we exploited the CRISPR/dCas9 system to manipulate the transcriptional level of target genes in common wheat. To improve target gene's expression, we generated transcriptional activator by fusing 6×TAL-VP128 activation domain to the C-terminus of dCas9 in frame. For target gene's repressing expression transcriptionally, 3×SRDX repression domain was conjugated to the C-terminus of dCas9 in frame. Our results showed that dCas9 fused activation or repression domain could increase or decrease the transcriptional level of target gene effectively in stable transgenic lines of wheat. The study on the tRNA-processing system in CRISPR/dCas9 based transcriptional regulation system demonstrated that this robust multiplex targeted tool can be incorporated to the CRISPR/dCas9 system to facilitate the target regulation of several genes' transcriptional level. Our data broaden the application of CRISPR/dCas9 based transcriptional regulation and provide great opportunities to investigate the function of essential genes in common wheat.
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Single-Molecule Imaging of Endogenous Proteins.
Single-molecule imaging is a technique of choice to investigate the dynamic nanoscale organization of proteins of interest, revealing a wealth of new information on how proteins perform their biological function. However, overexpression, which is often used in this context, can alter biological functions. It is therefore useful to implement tools to visualize and track endogenous proteins in living cells and observe their dynamic clustering and motion within their native environment. Herein, we describe two approaches: Fluorescent intrabody Localization Microscopy (FiLM) and gene-editing (CRISPR/Cas9) and provide a workflow including the design of plasmid backbones used to track endogenous proteins by either i) introducing nanobodies that target endogenous proteins to perform single-particle tracking or ii) gene-editing cells in culture to express tagged endogenous proteins at biologically relevant expression levels.
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Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins Results in High Levels of Gene Editing in Primary Hepatocytes.
Adeno-associated virus vectors are the most used delivery method for liver-directed gene editing. Still, they are associated with significant disadvantages that can compromise the safety and efficacy of therapies. Here, we investigate the effects of electroporating CRISPR-Cas9 as mRNA and ribonucleoproteins (RNPs) into primary hepatocytes regarding on-target activity, specificity, and cell viability. We observed a transfection efficiency of >60% and on-target insertions/deletions (indels) of up to 95% in primary mouse hepatocytes electroporated with Cas9 RNPs targeting Hpd, the gene encoding hydroxyphenylpyruvate dioxygenase. In primary human hepatocytes, we observed on-target indels of 52.4% with Cas9 RNPs and >65% viability after electroporation. These results establish the impact of using electroporation to deliver Cas9 RNPs into primary hepatocytes as a highly efficient and potentially safe approach for therapeutic liver-directed gene editing and the production of liver disease models.
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Activated macrophages of CD 163 gene edited pigs generated by direct cytoplasmic microinjection with CRISPR gRNA/Cas9 mRNA are resistant to PRRS virus assault.
Porcine reproductive and respiratory syndrome virus (PRRSV) infects placental and lung macrophages, causing a global epidemic with economic loss. Attempts to develop an effective vaccine to control the disease have not been effective. Currently, developing PRRSV disease-resistant pigs via a gene editing (GE) strategy to mutate the PRRSV receptor or to delete the binding domain on the macrophage appears promising. In this study, we used the strategy of Edinburg University to construct two guide RNAs (gRNAs) located on the proximal front and post sites of exon 7. Directive microinjection of two gRNAs and Cas9 mRNA into the cytoplasm of pronuclear zygotes efficiently generated four piglets confirmed as CD163 knockout (KO) and/or CD163 exon 7 deleted (CD163ΔE7). In four GE piglets, three pigs carried two chromosome CD163 KO or ΔE7. Peripheral blood mononuclear cells (PBMCs) from three GE and wild-type (WT) pigs were activated into macrophages for in vitro transfection. The results showed that the activated macrophages from all GE pigs were significantly more viable than those from WT pig. Current results suggest that we have successfully generated PRRSV-resistant pigs, although in vivo challenge is needed to validate that the pigs are PRRSV resistant.
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Antiretrovirals to CCR5 CRISPR/Cas9 gene editing - A paradigm shift chasing an HIV cure.
The evolution of drug-resistant viral strains and anatomical and cellular reservoirs of HIV pose significant clinical challenges to antiretroviral therapy. CCR5 is a coreceptor critical for HIV host cell fusion, and a homozygous 32-bp gene deletion (∆32) leads to its loss of function. Interestingly, an allogeneic HSCT from an HIV-negative ∆32 donor to an HIV-1-infected recipient demonstrated a curative approach by rendering the recipient's blood cells resistant to viral entry. Ex vivo gene editing tools, such as CRISPR/Cas9, hold tremendous promise in generating allogeneic HSC grafts that can potentially replace allogeneic ∆32 HSCTs. Here, we review antiretroviral therapeutic challenges, clinical successes, and failures of allogeneic and allogeneic ∆32 HSCTs, and newer exciting developments within CCR5 editing using CRISPR/Cas9 in the search to cure HIV.
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Functional Interrogation of Primary Human T Cells via CRISPR Genetic Editing.
Developing precise and efficient gene editing approaches using CRISPR in primary human T cell subsets would provide an effective tool in decoding their functions. Toward this goal, we used lentiviral CRISPR/Cas9 systems to transduce primary human T cells to stably express the Cas9 gene and guide RNAs that targeted either coding or noncoding regions of genes of interest. We showed that multiple genes (CD4, CD45, CD95) could be simultaneously and stably deleted in naive, memory, effector, or regulatory T cell (Treg) subsets at very high efficiency. Additionally, nuclease-deficient Cas9, associated with a transcriptional activator or repressor, can downregulate or increase expression of genes in T cells. For example, expression of glycoprotein A repetitions predominant (GARP), a gene that is normally and exclusively expressed on activated Tregs, could be induced on non-Treg effector T cells by nuclease-deficient Cas9 fused to transcriptional activators. Further analysis determined that this approach could be used in mapping promoter sequences involved in gene transcription. Through this CRISPR/Cas9-mediated genetic editing we also demonstrated the feasibility of human T cell functional analysis in several examples: 1) CD95 deletion inhibited T cell apoptosis upon reactivation; 2) deletion of ORAI1, a Ca2+ release-activated channel, abolished Ca2+ influx and cytokine secretion, mimicking natural genetic mutations in immune-deficient patients; and 3) transcriptional activation of CD25 or CD127 expression enhanced cytokine signaling by IL-2 or IL-7, respectively. Taken together, application of the CRISPR toolbox to human T cell subsets has important implications for decoding the mechanisms of their functional outputs.
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Optimized protocol to create deletion in adherent cell lines using CRISPR/Cas9 system.
The CRISPR/Cas9 system is a powerful tool for genome editing and is adaptable for a wide range of applications. Here, we have put together a step-by-step protocol for generating knockout cell lines (coding or non-coding region) using CRISPR/Cas9 tool. The protocol below has been tested on adherent cell lines such as HeLa and MCF7. However, it may easily be adapted to other adherent cell lines with minor variations. For complete details on the use and execution of this protocol, please refer to Farooq et al. (2021).
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Craspase: A novel CRISPR/Cas dual gene editor.
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Surprising CRISPR roadblocks.
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