Reliable, affordable, and quick detection of cancer-relevant mutations supports early diagnosis of patients with cancer and monitoring of treatment response or potential disease relapse. However, many current diagnostic methods rely on expensive, labor-intensive, and time-consuming next generation sequencing techniques to detect DNA changes in tumor cells. To improve cancer screening and monitoring, researchers at Cancer Center Amsterdam are developing a new diagnostic test that can detect cancer-specific DNA mutations quickly, accurately, and cost-effectively. Their findings were recently published in the CRISPR Journal.

The Nobel Prize-winning CRISPR technology has provided researchers with the molecular tools to precisely find a location in the genome of living cells and make changes in the DNA code. Over the last decade, CRISPR has fueled an unprecedented tsunami of scientific discoveries in molecular biology and shown promise in curing genetic diseases. In addition, the ability of CRISPR proteins to recognize a specific DNA sequence with great accuracy among the 6.4 billion other nucleotides of the human genome indicates the technology could be developed into an effective diagnostic tool for the detection of cancer-associated mutations.

Detecting Oncogenic Mutations in the Guardian of the Genome

“In this study, we show how to detect pathogenic mutations in the gene that encodes the master cell-fate regulator and guardian of the genome p53,” says Kavish Kohabir, PhD candidate at the Department of Human Genetics and first author of the publication. “Bacterial CRISPR proteins have been harnessed by synthetic biologists as reprogrammable molecular scissors that can cut DNA or RNA. We focused on CRISPR-associated protein Cas12a, a recently characterized CRISPR nuclease with exotic catalytic properties that are particularly suited for targeted genetic diagnostics.”

“Approximately half of all cancers have oncogenic mutations in TP53. The most abundant among these DNA changes affect arginine at position R273, making this an ideal candidate to establish a proof-of-principle diagnostic test for the detection of cancer-associated mutations.”

Kavish Kohabir, PhD student in Human Genetics at Amsterdam UMC.

Test development: identifying the right tools and methods

To develop a quick, sensitive, and specific test, the researchers applied PCR to amplify exon 8 of TP53 and tested various Cas12a ‘mutation detector’ proteins derived from different bacterial species. Kavish: ”We found that not every available Cas12a protein variant provides equivalent diagnostic fidelity. Also, we discovered that too much target pre-amplification triggers false-positive results – even with a high-fidelity polymerase.” Limiting DNA amplification means less input DNA to perform CRISPR mutation diagnostics and longer incubation times to achieve a positive read-out of the fluorescent reporter assay. This revealed a trade-off between specificity and test duration.

Towards Liquid Biopsy Testing of Oncogenic Mutations Using CRISPR

In a future diagnostic setting, the CRISPR test could potentially be used to detect oncogenic mutations in cell-free tumor DNA in blood or urine samples. “We simulated liquid biopsy tumor fractions from cultured wild type and mutant p53 cell lines and showed that our CRISPR-based diagnostic setup was able to reliably distinguish mutations and wild type TP53 DNA in under 2 hours,” Kavish explains.

Dr. Rob Wolthuis, principal investigator and last author of the publication, continues: ”The insights obtained in this study demonstrate that understanding Cas12a reaction dynamics can be used to greatly improve diagnostic performance to the single-nucleotide level. Now, we have established a proof-of-principle for an affordable, simple, and accurate diagnostic test for frequently occurring DNA mutations in cancer. Importantly, we performed accurate diagnostic tests on simulated liquid biopsy material. This shows our detection assay could be developed in a quick and affordable diagnostic targeted mutation test at the clinical point of care. This can improve tailored cancer screening and monitoring of individual cancer patients based on non-invasive blood or urine samples.”

For more information, contact Kavish Kohabir, or read the publication:

Kohabir, K.A.V., et al. (2023) In Vitro CRISPR-Cas12a-Based Detection of Cancer-Associated TP53 Hotspot Mutations Beyond the crRNA Seed Region. CRISPR Journal, https://doi.org/10.1089/crispr.2022.0077.

Prof. Jennifer Doudna (left), one of the Nobel laureates in Chemistry 2020 ‘for the development of a method for genome editing’. Kavish Kohabir (right), PhD student in Human Genetics at Amsterdam UMC

People involved at Cancer Center Amsterdam:

Kavish A.V. Kohabir

Lars O. Nooi

Arjen Brink

Ruud H. Brakenhoff

Erik A. Sistermans

Rob M.F. Wolthuis

Funding:

This study was partially funded by the Amsterdam UMC Innovation Grant.

Click this link for information about the Liquid Biopsy Center Amsterdam UMC – Cancer Center Amsterdam.

Do you want to apply CRISPR technology in your experiments? Contact the CRISPR Expertise Center at Cancer Center Amsterdam.

This article was created for Cancer Center Amsterdam.

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