CRISPR-mediated live cell imaging (Part 55- CRISPR in Gene Editing and Beyond)
Welcome to the 55th part of the multi-part series on applications of CRISPR in gene editing and beyond.
Fluorescence In Situ Hybridization (FISH)
Cytogenetic techniques like Fluorescence In Situ Hybridization (FISH) can detect and locate the precise location of particular DNA sequences on chromosomes. In this process, detecting the target DNA sequence can be compared to searching for a needle in a haystack, with the DNA sequence being the needle and the set of chromosomes being the haystack. The process is significantly facilitated by using a fluorescent probe that is complementary to the DNA sequence of interest. Therefore, the initial step in this process is to generate fluorescent probes through the nick translation technique. The probes used are generally short double-stranded DNA.
Nick Translation method
(i) The nick translation method uses the enzymes DNase I and DNA polymerase I. The enzyme DNAse creates random cuts or single-stranded nicks in one or both strands of the DNA to leave free 3' hydroxyl groups and 5’ phosphate termini. Now labeled nucleotides that have a fluorophore attached are provided. The free 3' hydroxyl group at the nicking site is used as a primer by the polymerase I enzyme. DNA polymerase I enzyme performs 2 activities:
· One is 5'-3' exonuclease activity that degrades DNA and removes the native nucleotides from free 5’ phosphate termini in the 5′→3′ direction (Fig 1a and b)
· and the other activity of DNA polymerase I enzyme is 5'-3' polymerase activity that uses free 3' hydroxyl group as a primer and the opposite strand as the template to synthesize a new strand complementary to the template (Fig 1c). The ligase enzyme then seals the gaps (Fig 1d).
Since the polymerase enzyme incorporates fluorescent nucleotides, the result is the replacement of unlabeled nucleotides with the labeled nucleotides in the ds DNA. This labeled DNA is called a probe.
(ii) The second step is the fixation of the cells with the formalin-based fixative that causes protein-protein and protein-DNA crosslinks. Then the fluorescent probes are added.
(iii) In the third step, the probe and chromosomal target DNA are denatured by heating the cell at 95°C. After that, the temperature is reduced, allowing the labeled probe to specifically bind to the target sequence (Fig 2). Once hybridization takes place, probes that are not bound to any DNA are washed away.
(iv) The results can be analyzed on a fluorescence microscope, which detects the fluorescent signals emitted from the labeled probe. The principle of fluorescence microscopy is based on the ability of certain molecules, such as fluorescent dyes or proteins, to absorb light of a specific wavelength and then emit light of a different wavelength. When a sample is illuminated with a specific wavelength of light, the fluorescent molecules in the sample absorb this light energy and become excited to a higher energy state. As the molecules return to their original energy state, they release the excess energy in the form of light of a longer wavelength. This emitted light is then captured by a camera or other detector, which converts the light into an image that can be analyzed by a computer or viewed by a human observer.
Components of a fluorescence microscope:
(i) Excitation Light Source: A light source (usually a high-intensity mercury lamp or a laser) emits light of higher energy and a shorter wavelength to excite the fluorescent molecules in the sample (Fig 3).
(ii) Excitation Filter: The excitation filter allows only the short wavelength of light to pass through it and removes the non-specific wavelengths of light.
(iii) Dichroic Mirror: The filtered light is then reflected by the dichroic filter and falls on the sample, which is fluorophore-labeled. The fluorophore molecules absorb shorter wavelength rays and get excited to a higher energy state. As the molecules return to their original energy state, they release the excess energy in the form of light of a longer wavelength.
(iv) Emission Filter: The emission filter blocks any residual excitation light and passes the desired longer emission wavelengths to the detector.
(v) Detector: A camera or other detector captures the emitted fluorescent light and forms glowing images of the fluorophore-labeled molecules against a dark background.
In the case of FISH, a fluorescent probe is used to label a specific DNA sequence of interest. The fluorophore attached to the probe emits a fluorescence signal when it is excited by the excitation light source. The emission light is then passed through the emission filter to the detector.
The specificity of the FISH probe is crucial for accurate results. If the probe is designed to bind only if it is 100% complementary to the gene and that sequence is present in the sample. In that case, the resulting image should show a bright spot of fluorescence corresponding to the location of the target DNA sequence. But if any sort of mutation occurred in this gene, no hybridization is possible, and no fluorescence signal will be detected at that location.
FISH technique can be used in the clinical diagnosis of various chromosomal abnormalities, including deletions, duplications, and translocations.
CRISPR-mediated live-cell imaging of genomic loci
Despite the widespread application, there are several drawbacks associated with FISH. First, the need for cell fixation makes the technique cumbersome for studying chromatin dynamics. Additionally, whether the state of chromatin architecture is properly preserved during FISH processing has always been questionable since the DNA duplex must be denatured to allow probes to hybridize with the target sequence.
The CRISPR-based imaging system is a recently-developed technology that has the potential to overcome the limitations of FISH and enable live-cell imaging of the genome. For this, dead Cas9 or dCas9 is used, which retains the ability to interact with sgRNA and to bind to target DNA. Either the dCas9 protein or sgRNA is modified to develop DNA imaging probes that integrate fluorescent proteins in a manner that doesn’t interfere with either dCas9 binding to sgRNA or sgRNA binding to the genomic sequence.
We have already discussed in Part 30 that dead Cas9 or dCas9 is a mutant in which both cleavage domains HNH and RuVC of Cas9 are inactivated. Although dCas9 can no longer cleave DNA, it can still bind target DNA with the same precision when guided by sgRNA.
Labeling dCas9 and sgRNA with fluorescent proteins
Let’s now understand how dCas9 and sgRNA can be labeled. The dCas9 can be labeled by fusing it with eGFP fluorescent protein. dCas9-eGFP, when introduced into the cell with sgRNA, can image the highly repetitive elements of the telomere region in the chromosome (Chen et al., 2013; Wu et al., 2019). The repetitive sequences can be imaged using a single gRNA, but imaging of nonrepetitive sequences requires simultaneous delivery of different gRNA per target (Fig 4).
For instance, the dCas9-eGFP protein has been used by scientists to image nonrepetitive regions of the MUC4 gene through the use of an array of at least 26 different sgRNAs.
To further improve genome imaging or gene detection, dCas9 can be fused with multiple fluorescent proteins through the use of the supernova tagging system. In this system, the dCas9 protein is modified to be linked with the SunTag, which is a repeating polypeptide array with multiple copies of GCN4 peptide (Fig 5). GCN4 peptide can recruit multiple copies of antibodies that are attached to a number of fluorescent proteins.
Alternatively to fused dCas9-fluorescent protein, a number of research groups have demonstrated the feasibility of imaging genome loci using modified sgRNAs that incorporate RNA hairpin aptamers. The aptamers can then recruit RNA-binding proteins fused to fluorescent proteins. The most widely used aptamer forms the binding sites for dimers of the bacteriophage MS2 coat proteins. MS2 coat proteins then can specifically bind to its cognate binding protein MCP fused to a fluorescent protein (Fig 6). Thus, each dCas9-sgRNA complex can then be tagged by multiple fluorescent proteins through MS2–MCP interactions (Tanenbaum et al., 2014; Ye et al., 2017).
A second approach, termed Casilio, employs the modification of sgRNA to harbor a unique 8-mer RNA sequence called PUF binding sequence or PBS (Cheng et al., 2016; Clow et al., 2020). Like MCP, PUF domain can also be fused to a fluorescent protein while retaining its capacity to bind to PBS (Fig 7). Engineering sgRNA with tandem repeats of PBS makes it possible to label the dCas9-sgRNA complex with multiple PUF fusion fluorescent proteins.
Advantages of CRISPR-mediated imaging
CRISPR-mediated imaging of cells offers several advantages over traditional imaging methods. Traditional imaging methods involve fixing cells or tissues and staining them with fluorescent probes. This process can alter the chromatin architecture during processing and limit the ability to study dynamic processes in live cells.
In contrast, CRISPR-mediated imaging of cells does not require cell fixation. Instead, the modified labeled dCas9 and gRNA are introduced into living cells using transfection or electroporation methods. The labeled dCas9 and gRNA then bind to the target DNA sequence and emit a fluorescent signal, allowing for real-time visualization of the genomic locus.
One significant advantage of this technique is the ability to study the dynamics of chromatin structure, gene expression, and other genomic processes in real-time. By observing changes in chromatin structure, gene expression, and other genomic events as they occur, researchers can better understand the dynamic mechanisms that regulate gene expression and genome function.
Another advantage of this technique is the ability to label multiple loci simultaneously using different fluorescent proteins. This approach enables researchers to study the interactions between different genomic regions and observe their dynamics in real-time.
Overall, CRISPR-mediated imaging of cells has the potential to significantly impact our understanding of the genome and its role in health and disease. Observing genomic events in real-time provides researchers with a powerful tool for understanding the complex processes that underlie gene expression and genomic function.
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References:
Cheng, A. W., Jillette, N., Lee, P., Plaskon, D., Fujiwara, Y., Wang, W., … & Wang, H. (2016). Casilio: a versatile CRISPR-Cas9-Pumilio hybrid for gene regulation and genomic labeling. Cell research, 26(2), 254–257.
Clow, P. A., Jillette, N., Zhu, J. J., & Cheng, A. W. (2020). CRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci. bioRxiv, 2020–03.
Wu, X., Mao, S., Ying, Y., Krueger, C. J., & Chen, A. K. (2019). Progress and challenges for live-cell imaging of genomic loci using CRISPR-based platforms. Genomics, proteomics & bioinformatics, 17(2), 119–128.
Chen, B., Gilbert, L. A., Cimini, B. A., Schnitzbauer, J., Zhang, W., Li, G. W., … & Huang, B. (2013). Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell, 155(7), 1479–1491.
Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S., & Vale, R. D. (2014). A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell, 159(3), 635–646.
Ye, H., Rong, Z., & Lin, Y. (2017). Live cell imaging of genomic loci using dCas9-SunTag system and a bright fluorescent protein. Protein & cell, 8(11), 853–855.
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