Conventional techniques of molecular diagnostics have gradually been falling short in terms of detection performance and accuracy.
However, the ability to recognize and cleave specific target sequences has made the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system an effective tool for molecular diagnosis of diseases such as cancer.
In a recent review published in LabMed Discovery, researchers from China discussed the use of CRISPR-Cas systems for the molecular diagnosis of tumor biomarkers, such as liquid biopsy biomarkers and tumor-specific gene variants and proteins. They explored the impact of CRISPR-Cas systems on molecular diagnostics.
Study: Advances in the applications of CRISPR/Cas system for tumor molecular diagnostics. Image Credit: Prostock-studio/Shutterstock.com
CRISPR-Cas Systems
Recent advances in molecular technology have moved the diagnosis and treatment of tumors into the realms of precision medicine, where molecular diagnostic tools and targeted therapy are widely used.
While conventional diagnostic methods, including polymerase chain reaction (PCR), can detect specific oncogenes and tumor proteins, the variability and complexity of new molecular biomarkers impact their sensitivity and accuracy.
The CRISPR-Cas system, which evolved as a defense mechanism in bacteria and archaea against phages and mobile genetic elements, has emerged as a versatile and powerful molecular diagnostic and therapeutic tool.
The CRISPR-Cas system uses a guide ribonucleic acid (RNA) to recognize specific gene sequences and direct the Cas proteins to cleave those sites — an ability that has been harnessed for the precise detection and editing of genes.
Tumor biomarker detection using CRISPR-Cas systems
The field of tumor diagnosis is increasingly moving to detecting molecular biomarkers such as gene and protein variants and liquid biopsy biomarkers for the precise diagnosis and treatment of cancers.
The CRISPR-Cas system is by itself, and in combination with other technologies such as aptamers, isothermal amplification, and nanomaterials, being widely used in cancer diagnostic platforms.
Some of the characteristics of CRISPR-Cas systems, such as the high specificity based on the precise base-pairing and use of the protospacer adjacent motif (PAM) in CRISPR-Cas9, have made it a valuable tool for tumor gene variant detection.
Rare mutations, such as those indicative of lung cancer, can be detected more precisely using CRISPR-Cas systems. Furthermore, technologies such as the CRISPR-Cas9-triggered hairpin probe have also been developed to counter the off-target effects of CRISPR-Cas9 and improve detection accuracy.
Conventional methods of protein detection, such as chemiluminescence and enzyme-linked immunosorbent assay (ELISA), struggle to detect proteins that are in low abundance, which is essential for the early detection of various cancers. CRISPR-Cas systems are being explored as an alternative method for the detection of low levels of proteins.
While the challenge has been to convert the recognition of the protein into a signal to activate CRISPR-Cas systems, methods such as strand displacement, proximity ligation, and nucleic acid self-assembly have proven successful in overcoming this obstacle.
The CRISPR-Cas13 system has been successfully used in a method that amplifies signals upon detection of vascular endothelial growth factor at sensitivity levels over a hundred times higher than that of ELISA.
The use of synthetic, highly specific nucleic acids known as aptamers along with CRISPR-Cas systems has also proven advantageous for the ultra-sensitive detection of tumor proteins such as carcinoembryonic antigen.
CRISPR-Cas-based systems such as Nano-CLISA and CRUISE have also made it possible to detect interferon-γ and epidermal growth factor receptors at levels as low as one femtogram per milliliter.
Additionally, CRISPR-Cas systems are being applied to detect liquid biopsy biomarkers. Liquid biopsies are non-invasive and focus on detecting exosomes and circulating tumor cells and tumor deoxyribonucleic acid (DNA) in body fluids, providing an opportunity for the early detection of cancers.
Strategies using CRISPR-Cas have been developed to detect proteins and micro RNAs inside exosomes that have been proven to be highly valuable for early cancer detection.
Furthermore, circulating tumor DNA, which carries tumor-related alterations, can be specifically targeted using CRISPR-Cas for ultra-sensitive detection and cancer monitoring.
CRISPR-Cas for in-situ Molecular Imaging
Understanding tumor heterogeneity is essential for the prediction of tumor progression, treatment outcomes, and drug resistance.
Tumors can be heterogeneous within and across patients, for which in-situ imaging and diagnosis are essential. Compared to conventional diagnostic methods, CRISPR-Cas is more effective in examining the heterogeneity within tumors without losing spatial information.
CRISPR-based imaging techniques, such as the dual-engineered CRISPR probe CoDEC and other Cas9 and Cas13-based systems, can help visualize single nucleotide variants and image RNA in tumor cells and simultaneously detect multiple messenger RNAs with high accuracy.
These methods have been used to detect the colocalization of breast cancer markers human epidermal growth factor receptor 2 (HER2), estrogen receptor, and progesterone receptor messenger RNAs.
Conclusions
To conclude, the use of CRISPR-Cas systems in the molecular diagnosis of tumors has significantly improved the accuracy and efficiency of tumor detection, especially for the early detection of tumors using low levels of biomarkers.
However, factors such as off-target effects continue to present challenges. The review highlighted the need for improving cancer detection by focusing on areas such as multiplex CRISPR-Cas-based diagnostics.
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