TAILIEUCHUNG - 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 .

• Sinh học
• Genome Biology
• Genome labeling
• Bimolecular fluorescence complementation
• Recruiting fluorescent proteins
• Genomic loci
• Pan genome
• Accessory genome
• Whole genome sequencing
• Next generation sequencing
• Genome wide association studies
• Synthetic biology
• Synthetic genomics
• Genome engineering
• Microbial evolution
• Laboratory evolution
• Summary of Doctoral Thesis in Biology
• Doctoral Thesis in Biology
• Analysis of mitochondrial genome
• Structure of mitochondrial genome
• Genetic characteristics of pig breeds
• Mango genome
• Whole genome duplication
• Notably cashew
• Family Anacardiaceae
• Genome resequencing clarifies
• Genome reconstruction
• Genome sequences
• Genome analysis
• Large scale genomic data sets
• Ancient DNA
• Mapping precision
• False positive rate
• Core genome
• Presence absence variation
• Genome assembly
• Population specific variation
• Base composition
• Genome divergence
• UV damage repair
• Plant domestication provides
• Genome structure
• Medfly genome
• Tephritid genomics
• Insect orthology
• Gene family evolution
• Chromosomal synteny
• Insect invasiveness
• Insect adaptation
• Genome surgery
• Genome editing
• Computational biology
• Distinguishing alleles
• High Yield cell and molecular biology
• Cell and molecular biology
• Chromosome replication
• Meiosis and genetic recombination
• The human nuclear genome
• The human mitochondrial genome
• Luận án Tiến sĩ
• Summary of Biology doctoral thesis
• Biology doctoral thesis
• Mitochondrial genome
• Northern provinces of Vietnam
• Functional redundancy
• Eukaryotic genome
• Escape selective constraints
• Genetic map
• K mer
• Genotypeby sequencing
• Chromosome scale genome assembly
• Ashkenazi human
• Human reference genome
• Single reference genome
• Population specific reference genomes
• Genome quality
• Genome contamination
• Metagenome assembled genomes
• Quantifies genome chimerism
• Genome graph
• Bacterial adaptability
• Human genome
• Transcriptional repression
• Genome higherorder organization
• Transcriptional activity
• Bacteriophage resistance
• CRISPR Cas9 genome editing tools
• Biological processes
• Genome sequence
• Automated circularization
• Genome assemblies
• Plasmodium falciparum
• Therapeutic genome
• Precisely modifying
• Infectious diseases
• 3D genome structure
• Higher order genome organization
• Population based modeling
• Homologous pairing
• Drosophila melanogaster
• Genome duplication
• Interspecific hybridization
• Genome evolution
• Regulatory elements
• Non coding regions
• Genome wide quantification
• Endogenous functionality
• Predictive modeling
• Synthetic organisms
• Multiplex genome engineering
• Hitchhiking mutations