Imaging genomic elements in living cells using CRISPR/Cas9.
In addition to their applications in genome editing and gene expression regulation, programmable DNA recognition systems, including both CRISPR and TALE, have been recently engineered for the visualization of endogenous genomic elements in living cells. This capability greatly helps the study of genome function regulation by its physical organization and interaction with other nuclear structures. This chapter first discusses the general considerations in designing and implementing the imaging system. The subsequent sections provide detailed protocols to use the CRISPR/Cas9 system to label and image specific genomic loci, including the establishment of expression systems for dCas9-GFP and sgRNA, the procedure to label repetitive sequences of telomeres and protein-coding genes, the simultaneous expression of many sgRNAs to label a nonrepetitive locus, and the verification of signal specificity by FISH.
Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA.
Baohui
B
Chen
Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA. Electronic address: bo.huang@ucsf.edu.
Bo
B
Huang
D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000032:analysis / Q000235:genetics; D064113:CRISPR-Cas Systems; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics; D015894:Genome, Human; D023281:Genomics / Q000379:methods; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D016615:Telomere / Q000235:genetics / Q000648:ultrastructure
D064130:CRISPR-Associated Proteins; D049452:Green Fluorescent Proteins
CRISPR; Chromatin; Fluorescence; Microscopy; Nucleus; Telomere