Affiliation:
1. Institute of Systems and Physical Biology Shenzhen Bay Laboratory Shenzhen China
2. Department of Biology, School of Life Sciences Southern University of Science and Technology Shenzhen Guangdong China
3. Alfred E. Mann Department of Biomedical Engineering University of Southern California Los Angeles California
Abstract
AbstractThe 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 constructsBasic Protocol 2: Transient transfection in mammalian cells and live‐cell imagingBasic Protocol 3: Generation of SIMBA‐expressing stable cell linesBasic Protocol 4: Manipulation of genomic loci using SIMBA
Funder
National Natural Science Foundation of China
Subject
Medical Laboratory Technology,Health Informatics,General Pharmacology, Toxicology and Pharmaceutics,General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Neuroscience