Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System

Abstract

SUMMARY The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of non-repetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.

Genome-wide identification of CRISPR/Cas9 off-targets in human genome

“…This might be largely due to additional involvement of the target sequence annealing step in activating the cleavage activities of CRISPR/Cas9 complex on its targets. On the other hand, the sgRNA-specific off-target binding activities may significantly affect other recently developed approaches which combine the nucleotide sequence binding specificity of CRISPR/Cas9 with other non-cleavage associated functions such as transcription regulation [14] and fluorescent labeling [15].…”

Developing bioimaging and quantitative methods to study 3D genome

“…For example, FISH was used to visualize the colocalization of olfactory receptor allele and an enhancer element in individual neurons [117]. CRISPR/Cas9-based technology [118,119] in conjuncted with 3C derivatives, can be used to study chromatin organization [120,121,122] in an elegant way. With the power of SRM, 3C-based methods and FISH/CRISPR technology are offering us more solutions to understand how the dynamics and organization of chromatin structure are functionally translated into the regulation of gene transcription, repair, and expression.…”

Genome editing: A breakthrough in life science and medicine [Review]

“…Inversely, fusion with an inactivation domain can be used to inactivate promoters [26]. Fusion with EGFP enables visualization of specific loci [27], and fusion with a tag protein enables chromatin immunoprecipitation of specific loci [28]. In combination with parallel on-chip gene synthesis, gRNA libraries for whole genes have been generated and used for functional screening [29], including screens for cancer-related genes [30].…”