Nano CRISPR-Cas9 gene a New Fast sequencing method for Cancer

In conventional genome sequencing, scientists must make many copies of the DNA they are trying to decipher, randomly break the DNA into segments, and feed the broken segments through a computerized machine that reads the string of nucleic acids. Then, 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.

Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods are limited by the loss of native modifications, short read length, high input requirements, low yield or long protocols.

Researchers at the biomedical engineering, molecular biology, and genetics at the Johns Hopkins University School of Medicine and led by Winston Timp were able to skip the DNA amplification step conventional sequencing 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 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 readout of DNA letters based on the unique electrical current that occurs when each chemical code “letter” slides through the hole. Findings from the new study were published recently in Nature Biotechnology.

Johns Hopkins scientists looked at 10 breast cancer genes and were able to use nanopore sequencing on breast cancer cell lines and tissue samples to detect DNA methylation. They 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 tumor spread. In the breast cancer cell lines, 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 tumor tissue taken at biopsies, the team was able to produce an average of 100 reads per region.

In addition to their studies of DNA methylation and small mutations, the investigators sequenced the gene commonly associated with breast cancer: BRCA1, which spans a region on the genome more than 80,000 bases long.

The study by Johns Hopkins scientists 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 and not another. Future studies underway to refine the CRISPR/nanopore sequencing technique and test its capabilities in other tumor types.

The researchers noted that pairing CRISPR with tools that sequence the DNA components of human cancer tissue is a technique that could, one day, enable fast, relatively cheap sequencing of patients’ tumors, streamlining the selection and use of treatments that target highly specific and personal genetic alterations.