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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: 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, Gene Editing, Jurkat T Cells, Lentivirus, dCas9, gRNA, IFNG-AS1
Experiment Summary: This protocol outlines a comprehensive procedure for CRISPR-based gene editing in Jurkat T cells using lentiviral vectors encoding dCas9 and guide RNAs (gRNAs). The experiment includes vector design and generation, virus generation and particle count, dCas9-VP64 transduction, selection, clone creation, and screening.
Experiment Tags: CRISPR-Cas9, Gene Editing, Lentivirus, AAV, Brown Adipose Tissue, Mouse Model
Experiment Summary: This Protocol optimizes CRISPR-Cas9 gene editing in brown adipose tissue (BAT) using lentivirus and AAV systems. Ucp1-Cre/Cas9 mice are used in in vivo research after screening effective sgRNAs in cultured cells and cloning them into AAV vectors. Western blot analysis is used to validate the effectiveness of gene deletion. By using this approach, the biology of BAT and its potential for treatment in metabolic diseases are better understood.
Experiment Tags: RMCE, Mouse Embryonic Stem Cells, Structure-Function Studies, Recombinase-Mediated Cassette Exchange, Gene Targeting, ROSALUC Mice, R26-iPSC Mice, pRMCE-DV1, Cre-Excised pRMCE-DV1, pCAG-FlpE-IRES-Puro-pA, Gateway pDONR221 Vector.
Experiment Summary: This protocol describes how to employ recombinase-mediated cassette exchange (RMCE) to perform structure-function research in mouse embryonic stem cells (mESCs). The process entails separating KO mESCs that are compatible with RMCE, creating a targeting vector that is compatible with RMCE, and using RMCE to target rescue constructs to the ROSA26 locus using mESCs.
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