UC researchers have discovered an unexpected side effect in the use of the CRISPR-Cas12a protein in DNA cutting that may potentially make disease diagnostics faster and more accurate.
Cas12a is a gene-cutting protein similar to the Cas9 protein that UC Berkeley professor of chemistry and molecular and cell biology Jennifer Doudna pioneered as a gene-editing tool. Unlike the Cas9 protein, however, Cas12a uses only a single nuclease to cut both strands of DNA and can cut double-stranded DNA at places that Cas9 cannot, according to Janice Chen, co-first author of the researchers’ report.
Researchers from the campus Doudna Lab, including co-first authors Chen, Enbo Ma and Lucas Harrington, were investigating the mechanism that allows a single nuclease in the Cas12a protein to cut the double-stranded DNA when they discovered the side effect.
“In doing these experiments, we came across the unexpected finding that once Cas12a binds to a matching target strand, the RuvC nuclease is activated to degrade any single-stranded DNA molecule, regardless of sequence,” Chen said in an email.
The researchers, along with Xinran Tian from UC Berkeley and Maria Da Costa and Joel Palefsky from UCSF, published their findings in the journal Science on Feb. 15.
In light of the discovery, the researchers realized that when Cas12a indiscriminately cuts all the single-stranded DNA, they could potentially detect specific sequences within the DNA, showing potential as a diagnostic tool.
“We developed a method called DETECTR, which uses Cas12a target recognition to cleave a single-stranded reporter molecule that emits fluorescence,” Chen said in an email. “This method can, in principle, be used to detect any DNA sequence of interest for point-of-care molecular diagnostics.”
To demonstrate the application of Cas12a, the researchers worked with Palefsky’s lab at UCSF and programmed the protein to detect human papillomavirus, or HPV, in real patient samples with high sensitivity and specificity.
According to Palefsky, the group chose to use HPV as an example because of its association with several human cancers, including cervical and anal cancer.
Chen said that while the new DNA detection method seems promising, the new activity may have unwanted side effects in terms of genome editing, and that more research is needed to find gene-editing applications.
The success of the DNA Endonuclease Targeted CRISPR Trans Reporter, or DETECTR, method under the HPV samples, however, shows promise in the use of Cas12a as a simple platform for molecular diagnostics.
“We are now exploring ways to expand the use of this technology to create a more broadly applicable test that could be used for clinical purposes,” Palefsky said in an email. “The ideal would be an inexpensive point-of care test that would make it particularly useful for cervical cancer screening in low resource settings where a test like this would be most needed.”