‘Cell Free’ CRISPR Could Improve Cancer Diagnostics
We are aware of the work researchers have been doing working on developing CRISPR gene editing as a potential therapeutic tool for a while now, but it is now being to use in cancer diagnostics.
According to Fierce Biotech, researchers from Christiana Care Health System used CRISPR to edit DNA outside the cell. This is a first in laying the groundwork for a diagnostic test that can replicate DNA mutations in patient’s tumour and help physicians identify the most appropriate treatment.
Team leader, Eric Kmiec, the director of Christina Care’s Gene Editing Institute, edited genes inside plasmids, a type of DNA that can be removed from a cell and manipulated in a lab dish. They initially started using Cas9, the backbone of many CRISPR systems, but it proved ineffective at editing DNA outside the cell. However, when they switched to a different enzyme, Cpf1 “it changed everything,” said Kmiec.
When Cas9 cuts DNA, it acts like a sharp pair of scissors, leaving “blunt ends,” added Kmiec. It requires precise positioning to insert replacement DNA because its ends are so smooth. In contrast, Cas12a leaves frayed, or “sticky,” ends. There are also single-stranded “DNA overhangs” that act like velcro, making it much easier to add DNA fragments to the sequence.
More often than not, when cancer patients are diagnosed, their tumours are often sequenced to identify genetic mutations that are drivers of their cancer. The CRISPR-Cas12a system can be used to replicate DNA mutations based on this sequencing information, Kmiec noted. Recreating them on synthetic pieces of DNA gives researchers a look at what pathways might be affected by mutations. Whereas a PCR-based method called site-directed mutagenesis already exists to recreate mutations in vitro, and using CRISPR could significantly accelerate the process.
In addition, the CRISPR-Cas12a system can also make multiple changes to DNA samples in one pass, while PCR systems require multiple rounds to replicate multiple mutations.
The team is now working on a “CRISPR-on-a-chip” for a commercial partner that has licensed the technology for cancer diagnostics. Despite diagnostics being the most immediate use of the technology, the Gene Editing Institute continues to work on therapeutic CRISPR with programs in lung cancer and melanoma. At present, CRISPR systems can only make edits on a single gene; down the line, Kmiec’s work could lead to a CRISPR therapy that treats diseases like Alzheimer’s that involve mutations in several genes.
This isn’t the first type of work being done with CRISPR for diagnostic purposes. Scientists over at the Broad Institute are developing a Cas13a-based system to test for Zika virus in blood, urine or saliva. The tool, known as SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing), targets RNA rather than DNA and doesn’t stop at cutting its intended target. Instead, it also cuts RNA that happens to be nearby, which the scientists branded “collateral cleavage.”
Another advantage of using CRISPR outside of the cell is that it can help demystify how, exactly, the technology makes its edits.
“When you’re working with CRISPR inside a cell, you’re kind of working in a black box where you can’t really observe the gears of the machinery that are doing these amazing things,” explained Kmiec. “You can see the results, the edits to the genes, but not necessarily how you got there, which is important for ensuring that CRISPR can be safely used to treat patients.”