In search of new ways to sequence human genomes and read critical alterations in DNA, researchers have successfully used the gene cutting tool CRISPR to make cuts in DNA around lengthy tumour genes – potentially informing future cancer treatment.
The researchers Johns Hopkins Medicine say this can be used to collect sequence information, and that pairing CRISPR with DNA sequencing tools, that sequence the DNA components of human cancer tissue, is a technique that could, one day, enable fast, relatively cheap, sequencing of patients’ tumours.
This could lead to streamlining the selection and use of cancer treatments that target highly specific and personal genetic alterations.
Tumour sequencing
In conventional genome sequencing, scientists have to make many copies of the DNA at issue. They then randomly break the DNA into segments, and feed the broken segments through a computerised machine that reads the string of chemical compounds called nucleic acids, made up of the four “bases” that form DNA, and are lettered A, C, G and T. Following this, scientists look for overlapping regions of the broken segments and fit them together like tiles on a roof to form long regions of DNA that make up a gene.
Winston Timp, PhD, assistant professor of biomedical engineering and molecular biology and genetics at the Johns Hopkins University School of Medicine, said: “For tumour sequencing in cancer patients, you don’t necessarily need to sequence the whole cancer genome. Deep sequencing of particular areas of genetic interest can be very informative.”
In their experiments, Timp and PhD student Timothy Gilpatrick were able to skip the DNA-copying part of conventional sequencing by using CRISPR to make targeted cuts in DNA isolated from a sliver of tissue taken from a patient’s breast cancer tumour.
Then, the scientists glued so-called “sequencing adaptors” to the CRISPR-snipped ends of the DNA sections. The adaptors serve as a kind of handle that guide DNA to tiny holes or “nanopores” which read the sequence.
By passing DNA through the narrow hole, 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.
Informing cancer treatment
Among 10 breast cancer genes the team focussed on, the Johns Hopkins scientists were able to use nanopore sequencing on breast cancer cell lines and tissue samples to detect a type of DNA alteration called methylation, where chemicals called methyl groups are added to DNA around genes that affect how genes are read.
The researchers found a location of decreased DNA methylation in a gene called keratin 19 (KRT19), which is important in cell structure and scaffolding. Previous studies have shown that a decrease in DNA methylation in KRT19 is associated with tumour spread.
In the breast cancer cell lines they studied, the Johns Hopkins team was able to generate an average of 400 “reads” per basepair, a reading “depth” hundreds of times better than some conventional sequencing tools.
Among their samples of human breast cancer tumour tissue taken at biopsies, the team was 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,” says Timp.
In addition to their studies of DNA methylation and small mutations, Timp and Gilpatrick sequenced the gene commonly associated with breast cancer: BRCA1, which 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,” says Gilpatrick.
Timp 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.”
In addition to its potential to guide treatment for patients, Timp says the combination of CRISPR technology and nanopore sequencing provides such depth that it may help scientists find new disease-linked gene alterations specific to one allele (inherited from one parent) and not another.
Timp and Gilpatrick plan to continue refining the CRISPR/nanopore sequencing technique and testing its capabilities in other tumour types.
