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Updated: Feb 12, 2026

Genome Editing in Mammalian Cell Lines using CRISPR-Cas
Published on: April 11, 2019
Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imaging
Yu Hong1,2, Guangqing Lu3,2, Jinzhi Duan3,2
1Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program, School of Life Sciences, Peking University, Beijing, 100871, China.
Abstract:
CRISPR/dCas9 binds precisely to defined genomic sequences through targeting of guide RNA (gRNA) sequences. In vivo imaging of genomic loci can be achieved by recruiting fluorescent proteins using either dCas9 or gRNA. We thoroughly validate and compare the effectiveness and specificity of several dCas9/gRNA genome labeling systems. Surprisingly, we discover that in the gRNA-labeling strategies, accumulation of tagged gRNA transcripts leads to non-specific labeling foci. Furthermore, we develop novel bimolecular fluorescence complementation (BIFC) methods that combine the advantages of both dCas9-labeling and gRNA-labeling strategies. The BIFC-dCas9/gRNA methods demonstrate high signal-to-noise ratios and have no non-specific foci.
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