FRONTIERS IN MEDICAL CASE REPORTS - Volume 4; Issue 4, (Jul-Aug, 2023)

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CRISPR/Cas9-Based Imaging: Challenges and Future Prospects

Author: Mahintaj Dara

Category: Medical Case Reports

Abstract:

In 1987, CRISPR (clustered regularly interspaced short palindromic repeats), was discovered in the Escherichia coli genome. It is a component of the adaptive immune system in prokaryotic, archaeal, and bacterial organisms. since it was discovered in bacteria, understanding CRISPR structure and function has made it a powerful gene editing tool that used in gene therapy and diagnostics purpose. Imaging is another application of CRISPR that is also used by researchers for diagnostic purposes. This short communication aims to discuss the CRISPR imaging field and the challenges facing it.

Keywords: CRISPR, Imaging, d Cas9, gRNA

DOI URL: https://dx.doi.org/10.47746/FMCR.2023.4401

Full Text:

Short Communication

Clustered regularly interspaced short palindromic repeats (CRISPR) that are part of an adaptive immune system in prokaryotic organisms, archaea, and bacteria, firstly discovered in the Escherichia coli genome in 1987 (Wang et al., 2022). According to the CRISPR system, after the first attack of the phage on the bacteria, the foreign gene fragments of the phage insert into the bacterial genome and memorize the genetic information of the foreign species (LaFleur et al., 2019). These unique sequences are interspersed within the host array of short repetitive DNA sequences, creating Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). The CRISPR region transcribes a small piece of RNA or guide RNA (gRNA) that contains the genetic information of the foreign DNA (Phage DNA) and translates it into a CRISPR-associated protein (Cas) with endonuclease activity. After a second attack on the phage, the Cas and gRNA are complexed and cleave the same phage DNA using the genetic information stored in the genome (LaFleur et al., 2019). Since the discovery of the CRISPR system in bacteria, understanding of CRISPR structure and function has made it a powerful gene editing tool used in gene therapy, and diagnostics fields (Liu et al., 2021). Promising results are shown for gene therapy of genetic diseases such as sickle cell anemia, thalassemia, Duchene muscular dystrophy, leukemia, and even types of solid tumors using CRISPR technology (Rheney, 2023; Choi and Koo, 2021; Vuelta et al., 2021; Tang et al., 2020). Although some of these studies are in the research phase, others, such as gene therapy for sickle cell anemia, have entered the clinical trial phase (Anliker et al., 2022).

Moreover, the CRISPR system has been highly successful and effective in detecting infectious and non- infectious diseases so far that it could be used as a replacement for currently available laboratory diagnostic tests (Kim et al., 2021). Today, CRISPR has attracted the attention of researchers as a tool for imaging DNA and genomic loci of interest (Lucansky et al., 2023).

For imaging with CRISPR technology, used a modified Cas9 protein without cleavage activity named dCas9. The dCas9 has been tagged with an enhanced green fluorescent protein, EGFP, and a particular gRNA is designed for a target sequence (Selvaraj et al., 2021). The modified Cas9 and gRNA were introduced into cells. If the desired genomic locations are present in the sample or cell, a fluorescent signal is emitted when the guide RNA/dCas9 complex binds to the target sequence, and the operator or researcher can detect EGFP inside the cells (Selvaraj et al., 2021) (Fig. 1). This innovative work makes it possible to see cells at a high resolution using CRISPR imaging, providing scientists with new ways of understanding cell dynamics (Wu et al., 2019). Research efforts about CRISPR imaging are divided into two main groups due to its relatively new technology: chromatin remodels and telomere health (Wu et al., 2019; Thuma et al., 2023). Although many advances have been made in the field of CRISPR imaging, several challenges remain. To improve this technology, many efforts have been made to find solutions to the described challenges (Wu et al., 2019). The first challenge is to correctly identify the signal emitted from the sample, which can cause false positive results because background intensity and non-specific signals can affect the results (Thuma et al., 2023). To reduce and overcome this problem, the transfection method should be optimized. The use of CRISPR imaging is mainly used to image repetitive sequences of the genome. This is despite the fact that a small percentage of the genome consists of repetitive sequences. This problem limits the application of CRISPR imaging (Mao et al., 2019). To enhance the use of CRISPR imaging to target non-repetitive sequences, one solution is to use multiple sgRNAs that target sites flanking the target sequence (Lyu et al., 2022). Unwanted binding of gRNA to non-specific regions (off-targets) is one of the important challenges that the CRISPR system faces in both editing and detection as well as imaging (Chaudhary et al., 2021). Despite careful consideration during gRNA design, the off-target occurrence is inevitable. Overcoming this important challenge can increase the application and use of the CRISPR system in various fields of treatment, diagnosis, and imaging.

Conclusion

In addition to diagnosis and gene therapy, the CRISPR system can also be used successfully in the field of imaging.

Figure 1: Schematic figure of CRISPR imaging technology. dCas9 that tagged to an enhanced green fluorescent protein (EGFP) guided to the target sequence of the genome by gRNA. The modified Cas9 and gRNA were introduced into cells and the researcher was able to detect EGFP inside the cells.

Conflict of Interest: Mahintaj Dara declares that she has no conflict of interest.

Ethics Approval: Not applicable.

Consent to Participate: Not applicable.

Consent for Publication: Not applicable.

Availability of Data and Material: The datasets collected during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding: This study supported by Shiraz University of Medical science.

Authors' Contributions: Mahintaj Dara participated in the study conception and design, data analysis, manuscript revision, and approval of the final content of the manuscript. The author read and approved the final manuscript.

Acknowledgments: The author would like to thank Ms. Firoozeh. Dara for improving the use of English in the manuscript

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