Targeted Gene Cuts Could Accelerate Cancer Tumor Sequencing

Combining the gene cutting tool CRISPR with tools that sequence the DNA components of human cancer tissue could lead to fast, relatively cheap tumor sequencing for cancer patients, according to a study published in Nature Biotechnology.

Researchers at Johns Hopkins Medicine used CRISPR to make cuts in DNA around lengthy tumor genes, which could help collect sequencing information. The technique could streamline the selection and use of treatments that target highly specific and personal genetic alterations.

“For tumor sequencing in cancer patients, you don't necessarily need to sequence the whole cancer genome,” said Winston Timp, PhD, assistant professor of biomedical engineering and molecular biology and genetics at the Johns Hopkins University School of Medicine. “Deep sequencing of particular areas of genetic interest can be very informative.”

In traditional genome sequencing, scientists have to make many copies of the DNA, randomly break the DNA into segments, and feed the broken segments through a machine that reads the nucleic acids, which are made up of the four bases that form DNA and are labeled A, C, G, and T. Scientists then look for overlapping regions of the broken segment and fit them together to form long regions of DNA that make up a gene.

In the study, researchers were able to skip the DNA-copying aspect by using CRISPR to make targeted cuts in DNA isolated from a sliver of tissue taken from a patient’s breast cancer tumor. Then, the team glued sequencing adapters to the CRISPR-cut ends of the DNA sections. The adapters guide the DNA to tiny holes or nanopores that read the sequence.

By passing DNA through the narrow holes, a sequencer can build a read-out of DNA letters based on the unique electrical current that occurs when each chemical code letter slides through the hole.

Among the ten breast cancer genes the researchers focused on, Johns Hopkins scientists were able to use nanopore sequencing on breast cancer cell lines and tissue samples to identify a type of DNA alteration called methylation, where chemicals called methyl groups are added to DNA around genes and affect how genes are read.

Researchers found a location of decreased DNA methylation in a gene called keratin 19 (KRT19), which is important in cell structure and scaffolding. Previous research has shown that a decrease in DNA methylation in KRT19 is associated with tumor spread.

In the breast cancer cell lines they studied, the team was able to generate an average of 400 reads per base pair, which is a reading depth one hundred times better than some traditional sequencing tools.

Among their samples of human breast cancer tumor tissue taken at biopsies, researchers were able to produce an average of 100 reads per region.

“This is certainly less than what we can do with cell lines, but we have to be more gentle with DNA from human tissue samples because it’s been frozen and thawed several times,” said Timp.

Researchers also sequenced the BRCA1 gene, which is commonly associated with breast cancer. BRCA1 spans a region on the genome more than 80,000 bases long.

“This gene is really long, and we were able to collect sequencing reads which went all the way through this large and complex region,” said Timothy Gilpatrick, MD/PhD student.

The new method could significantly improve DNA sequencing for cancer tumors, researchers said. 

“Because we can use this technique to sequence really long genes, we may be able to catch big missing blocks of DNA we wouldn’t be able to find with more conventional sequencing tools,” said Timp.

The team plans to further refine the CRISPR and nanopore sequencing technique and test its abilities in other types of tumors.