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换一换Production of CRISPR-Cas9 Transgenic Cell Lines for Knocksideways Studies
Michael Wagenbach, Juan Jesus Vicente, Wren Wagenbach, Linda Wordeman, Michael Wagenbach, Juan Jesus Vicente, Wren Wagenbach, Linda Wordeman
Protein activity is generally functionally integrated and spatially restricted to key locations within the cell. Knocksideways experiments allow researchers to rapidly move proteins to alternate or ectopic regions of the cell and assess the resultant cellular response. Briefly, individual proteins to be tested using this approach must be modified with moieties that dimerize under treatment with rapamycin to promote the experimental spatial relocalizations. CRISPR technology enables researchers to engineer modified protein directly in cells while preserving proper protein levels because the engineered protein will be expressed from endogenous promoters. Here we provide straightforward instructions to engineer tagged, rapamycin-relocalizable proteins in cells. The protocol is described in the context of our work with the microtubule depolymerizer MCAK/Kif2C, but it is easily adaptable to other genes and alternate tags such as degrons, optogenetic constructs, and other experimentally useful modifications. Off-target effects are minimized by testing for the most efficient target site using a split-GFP construct. This protocol involves no proprietary kits, only plasmids available from repositories (such as addgene.org). Validation, relocalization, and some example novel discoveries obtained working with endogenous protein levels are described. A graduate student with access to a fluorescence microscope should be able to prepare engineered cells with spatially controllable endogenous protein using this protocol. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. **Basic Protocol 1** : Choosing a target site for gene modification **Basic Protocol 2** : Design of gRNA(s) for targeted gene modification **Basic Protocol 3** : Split-GFP test for target efficiency **Basic Protocol 4** : Design of the recombination template and analytical primers **Support Protocol 1** : Design of primers for analytical PCR **Basic Protocol 5** : Transfection, isolation, and validation of engineered cells **Support Protocol 2** : Stable transfection of engineered cells with binding partners
AI 解读Single Genomic Loci Labeling and Manipulation Using SIMBA System
Yanwei Chen, Ziliang Huang, Qin Peng, Yingxiao Wang, Yanwei Chen, Ziliang Huang, Qin Peng, Yingxiao Wang
The SIMBA (Simultaneous Imaging and Manipulation of genomic loci by Biomolecular Assemblies) system is an innovative CRISPR-based imaging technique that leverages dCas9-SunTag and FRB-mCherry-HP1α, with scFv-FKBP acting as a bridge. This powerful system enables simultaneous visualization and manipulation of genomic loci. The dCas9-SunTag fusion protein allows for precise targeting of specific genomic sites, and the FRB-mCherry-HP1α fusion protein facilitates the condensation of chromatin at the targeted loci. The scFv-FKBP bridge protein links dCas9-SunTag and FRB-mCherry-HP1α, ensuring efficient and specific recruitment of HP1α to the desired genomic loci. This integrated approach allows us to visualize and manipulate genomic regions of interest, opening up new avenues for studying genome organization, gene expression regulation, and chromatin dynamics in living cells. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. **Basic Protocol 1** : Cloning of genetic constructs **Basic Protocol 2** : Transient transfection in mammalian cells and live-cell imaging **Basic Protocol 3** : Generation of SIMBA-expressing stable cell lines **Basic Protocol 4** : Manipulation of genomic loci using SIMBA
AI 解读Bulk FLASH-seq
Rebecca A Siwicki, Vincent Hahaut, Simone Picelli
Bulk RNA sequencing has revolutionized the study of transcriptomes, enabling the analysis of gene expression in complex tissues and heterogenous cell populations. While single cell RNA sequencing (scRNA-seq) has gained popularity due to its ability to profile individual cells, it comes with limitations such as high costs and reduced sensitivity for detecting low-abundance transcripts. Here, we present bulk FLASH-seq (FS), a full-length RNA-seq method based on the single cell FLASH-seq workflow (Hahaut _et al_ , 2022, [https://www.nature.com/articles/s41587-022-01312-3](https://www.nature.com/articles/s41587-022-01312-3)), updated for bulk RNA analysis. FS bulk generates high quality data while requiring minimal hands-on time and offering a greater degree of customization. As a homebrew protocol, it is inexpensive compared to commercial kits allowing you to invest in greater sequencing depths or in a higher number of sequenced samples. Our protocol enables comprehensive transcriptome analysis of bulk RNA samples, providing an alternative approach to scRNA-seq for gene expression when single-cell RNA-sequencing is not required.
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