tailieunhanh - Comparison and optimization of CRISPR/ dCas9/gRNA genome-labeling systems for live cell imaging

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. | Hong et al. Genome Biology 2018 19 39 https s13059-018-1413-5 METHOD Open Access Comparison and optimization of CRISPR dCas9 gRNA genome-labeling systems for live cell imaging Yu Hong1 3 Guangqing Lu2 3 Jinzhi Duan2 3 Wenjing Liu2 3 and Yu Zhang1 2 3 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. Keywords Genome labeling CRISPR dCas9 Bimolecular fluorescence complementation BIFC Background in live cells. In its first version direct fusion of fluorescent The dynamic localization of a particular genomic locus proteins such as green fluorescent protein GFP with in a three-dimensional 3D genome has been proposed dCas9 protein was used by Huang s laboratory 11 . To in- to regulate various genome functions including gene crease the signals a SunTag that contains multiple copies transcription DNA recombination DNA replication 24X of GCN4 peptide epitopes has been added to the C- and DNA repair 1 2 . Until recently several strategies terminal dCas9 12 . Fusion with single-chain fragment have been developed to trace the dynamic movement of variable scFv antibody against GCN4 peptide allows genomic loci in living cells 3 . Clustered regularly inter- more copies of fluorescent proteins to be recruited to a spaced short palindromic repeats CRISPR CRISPR- single tethered dCas9 .