Shining Light on the Genome with CRISPR

Real-time cellular imaging with CRISPR-Cas9

CRISPR-based gene-editing technologies have revolutionized biological research by allowing the quick and easy targeted editing of genetic sequences, supporting a myriad of applications such as the creation of engineered cell lines and animal models as well as functional genomic screening. But scientists have also looked to adapt this versatile system to applications beyond gene editing—CRISPR-Cas9 can also be used to localize fluorescent probes to specific sites in the genome and thus employed as a real-time cellular imaging tool. This article looks at how the development of the CRISPR-Cas9 system can improve visualization and understanding of cellular processes, such as chromatin architecture and cellular relationships between genes and proteins.

The challenges of spatiotemporal imaging

An organism’s response to its environment is coordinated by careful regulation of gene expression. Key to this is the spatiotemporal organization of chromatin, which plays a crucial role in regulating genome function, facilitating transcription, differentiation, and development. Having an accurate representation of this dynamic interplay is crucial to furthering our understanding of the complex cellular dynamics that govern gene expression and cell behavior. However, visualizing interactions between specific molecules in real space and time is difficult. Traditional methods of imaging nucleic acids usually require sample fixation and DNA denaturation, such as fluorescence in situ hybridization (FISH), and so are incompatible with live imaging. The use of fluorescently tagged DNA-binding proteins, for example TALEN-based systems, can be been employed for live-cell imaging but are complex to design and implement, requiring a high level of expertise in protein engineering, and cannot bind non-repetitive sequences that make up a large proportion of the genome, including many important regulatory regions.

CRISPR-Cas9: more than a gene editor

Since its debut in 2012, the ease of design and implementation of the CRISPR-Cas9 system as well as its inherent flexibility—the Watson and Crick base pairing of the short guide RNA (sgRNA) with the target sequence means that it can recruit the Cas9 protein to almost any genomic loci—has led to its rapid adoption as a gene-editing tool. Wild-type Cas9 is commonly used to knockout gene function through disruption of the coding sequence, but a catalytically inactive version of Cas9 (dCas9) can also be used to bring different functional domains to the target site, such as transcriptional activators or repressors. This has resulted in the continual expansion of the CRISPR toolkit, with a range of perturbations now possible beyond knockout, such as changes to gene expression with CRISPRi and CRISPRa, and even base editing. It was therefore not long until researchers looked to use dCas9 to recruit fluorescent domains to the target site. In their seminal paper in 2013, Chen et al successfully targeted dCas9 with eGFP to image dynamic behaviors of telomeres and coding genes during mitosis in living cells. In addition, they also showed that by using an array of sgRNA tiled across a target locus, they could even visualize non-repetitive sequences.

CRISPR as an imaging tool

The spatiotemporal control of 3D genomic architecture has a profound impact on phenotype, and so CRISPR-based imaging, unlike previous technologies, can be used to probe and monitor chromatin dynamics in real-time in a native cellular context. Given that chromatin remodeling is key to gene expression, as it is ubiquitous in almost every biological process, understanding the dynamics of this mechanism is crucial.

CRISPR-based imaging is still in the early stages of development, and so there are still limitations to overcome—such as improving the efficacy of dCas9 targeting due to sequence and chromatin-based constraints and improving the signal to noise ratio. But like many CRISPR developments, the advancements in CRISPR-based imaging have continued. The initial study demonstrated that non-repetitive regions can be imaged but this required at least 26 sgRNA tiled across the region of interest for sufficient signal to be detected. To improve labeling efficiency within non-repetitive regions, several approaches have been utilized, including use of the SunTag system, a repeating peptide array that can recruit multiple copies of eGFP. Chaudhary et al also used the SunTag system with tripartite fluorescent proteins to develop a system that strongly suppressed background fluorescence.

More recently, Mao et al used molecular beacons that undergo fluorescence resonance energy transfer (FRET) to develop a CRISPR/dual-FRET MB system to dynamically track non-repetitive loci with only three sgRNA. Multi-color imaging has also been developed by employing two different Cas9 proteins in a combinational approach—the most commonly used Cas9 from S. pyogenes and an alternative from S. aureus, thereby allowing two-color imaging, enabling visualization of multiple genomic loci in the same cell.

As well as visualizing DNA, work has also turned to using CRISPR to monitor the location and dynamics of RNA molecules in living cells. Yang et al used dCas13 to demonstrate robust labeling of several RNAs within living cells—and also that orthogonal dCas13 proteins and/or dCas9 systems allowed the simultaneous detection of genomic DNA and RNA transcripts in real-time. A similar approach was utilized by Wang et al, who developed the LiveFISH system, an approach that uses fluorescent oligos for genome imaging even in primary cells.

The future is bright

CRISPR-based imaging is enabling the visualization of 3D genomic architecture in real-time in living cells, offering a powerful tool to aid our understanding of cellular processes and those that underpin disease states. Dynamic biochemical events such as chromosome behavior during cell differentiation, translocations, DNA repair, or interactions between promoters/enhancers that drive disease phenotypes could be probed and quantitatively assessed at the molecular level in a single cell. This is yet another example of the versatility of the CRISPR system—and given how revolutionary CRISPR applications have been and how new this approach is, it is highly likely that the full impact of CRISPR-based imaging on our understanding of biology is yet to come.

Key Takeaways

• As well as gene editing, the CRISPR system has been used to recruit fluorescent probes to specific genomic targets, therefore enabling live-cell imaging in real-time.
• Unlike existing technologies such as FISH and TALEN-based imaging, DNA imaging with CRISPR can be performed in living cells, does not require labor-intensive protein engineering, and can be used to track non-repetitive sequences that make up a large proportion of the genome.
• CRISPR-based imaging has been used to visualize chromatin dynamics and monitor telomere health, which play a fundamental role in biological processes and disease.