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Experiment Tags: sgRNA Design, CRISPR-Cas9 System, sgRNA Library, Gene Editing, Gene Regulation
Experiment Summary: In this protocol, we discusse how to design sgRNA sequences and genome-wide sgRNA library using CRISPR-ERA.
Experiment Tags: CRISPR/Cas9, AAV (AAV), Adult Mouse Brain, Gibson Assembly, Dual-sgRNA
Experiment Summary: In this protocol, we describe a detailed protocol of AAV vector construction for cell type-specific CRISPR gene editing in the adult mouse brain. The method adopts a dual-sgRNA strategy for efficient disruption of the target gene. At first, a few different sgRNAs targeting the same gene are cloned into a plasmid expressing spCas9. After evaluation of the sgRNAs by a T7 endonuclease assay, the two most efficient sgRNAs are cloned in tandem into an AAV vector using the Gibson Assembly method.
Experiment Tags: T-box (T-box), EMSA (EMSA), RNA-RNA Interaction, TRNA (tRNA), In Vitro Transcription
Experiment Summary: This protocol describes the coupling of (i) “live” in vitro RNA transcription with (ii) binding by a radiolabeled, pre-formed tRNA followed by native gel electrophoresis and phosphorimager scan to visualize the complex.
Experiment Tags: Nucleoside-Modified mRNA, Lipid Nanoparticle, Liver Regeneration, Protein Expression, Retro-orbital Injection
Experiment Summary: In this protocol, we describe in detail the necessary steps to deliver mRNA-LNP to the murine liver and, following intravenous injection of eGFP mRNA-LNP, verify transfection efficiency using flow cytometry and liver cell specificity using immunofluorescence analyses.
Experiment Tags: Recombinant Virus, Vaccinia Virus, VACV (VACV), CRISPR/Cas9 Selection, Virotherapy, Vaccine Development
Experiment Summary: In this protocol, we describe protocols for the generation and enrichment of recombinant vaccinia viruses using targeted Cas9 as a selection tool. This novel use of Cas9 is a simple addition to current homologous recombination-based methods that are widespread in the field, facilitating implementation in laboratories already working with poxviruses.
Experiment Tags: dsRNA Synthesis, In Vitro Preparation
Experiment Summary: In this protocol, we describe a simple and widely used in vitro transcription reaction for the preparation of double-stranded RNA using PCR templates. The dsRNA prepared by this method can be used to induce RNA interference in certain cells or tissues.
Experiment Tags: CRISPR/Cas9 (CRISPR/ Cas9), Adeno-Associated Viral Vector, Trans-Zona Pellucida, Intra-Embryo Genome Editing, Ribonucleoprotein Electroporation, Large Fragment Knock-In
Experiment Summary: In this protocol, we demonstrate a detailed protocol for AAV-mediated intra-embryo knock-in in laboratory mice. This technique does not require either micromanipulation or germline competent pluripotent stem cells.
Experiment Tags: Nucleoside-Modified mRNA, Lipid Nanoparticle, Liver Regeneration, Protein Expression, Retro-Orbital Injection
Experiment Summary: In this protocol, we describe in detail the necessary steps to deliver mRNA-LNP to the murine liver and, following intravenous injection of eGFP mRNA-LNP, verify transfection efficiency using flow cytometry and liver cell specificity using immunofluorescence analyses
Experiment Tags: CRISPR-Cas9, Off-Target Activity, Chromosomal Translocation, LAM-HTGTS, iHTGTS
Experiment Summary: In this article, we described an improved HTGTS (iHTGTS) method, in which size-selection beads were used to enhance reaction efficiency and a new primer system was designed to be compatible with Illumina Hiseq sequencing. iHTGTS is a powerful method for comprehensively assessing Cas9 off-target effect.
Experiment Tags: Diagnosis, RNA Virus, Replicative Intermediate, dsRNA Isolation, dsRNA-Binding Protein
Experiment Summary: In this protocol, we describe a method for dsRNA isolation. Using this method, we can readily isolate viral dsRNA from a small amount of plant material, and can process numerous samples simultaneously.
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