Researchers at the Department of Biomedical Engineering at Johns Hopkins University have successfully created a new and revolutionary method to collect and study gene samples. They accomplished this by using the gene cutting tool CRISPR to retrieve sections of DNA that were then studied using nanopores. They have used this new information to study genetic information from the human breast cancer gene BRCA1. They have also used this method to determine concentrations of the chemical group methyl on DNA molecules. Thanks to this new method, researchers will no longer have to study the entire genome to find gene of interest.
The CRISPR/nanopore tools could revolutionize the process of creating readable sequences of human cancer tissue DNA. In the future, this technique could become a fast and cheap means of studying genetic data from the tumors of cancer patients. This could allow us to create highly targeted genetic treatments that could even be used to induce genetic changes in patients. This technique also allows us to catch large blocks of DNA that go missing when we use previously established methods.
The CRISPR gene cutting tool was created using the Cas 9 gene, which is naturally found in bacteria. Cas 9 behaves like a pair of scissors and is used to cut and retrieve sections of DNA. In the study, the researchers took cut sections of DNA and glued them to “sequencing adaptors”, which are tools that pass the DNA through nanopores.
Nanopores are tiny holes that the DNA can be passed through. By passing sections of DNA through these holes, the researchers were able to read the sequence of the molecules that make up the DNA section based on the unique electrical signal that occurs when each molecule goes through the hole.
The researchers then studied the degree of methylation of DNA sections from cancer DNA and non-cancer DNA. The CRISPR/nanopore method allowed the researchers to accurately characterize and study the frequency of genetic differences between the cancerous and non-cancerous DNA sections. A decrease in DNA methylation has been found to be associated with tumor spread. The researchers discovered similar decreased DNA methylation in the KRT19 gene found in the cancer DNA.
Using the CRISPR/nanopore method, the researchers studied tissue samples from normal human breast tissue and a breast cancer cell line derived from a tumor. This allowed the researchers to achieve a reading of the BRCA1 gene that spanned an area of 80,000 single molecules that collectively make up DNA. In the breast cancer cell line that was studied, the researchers were able to create a reading that had an average of 400 ‘reads’ per single molecule. A depth that is hundreds of times more accurate than previous methods being used.
In prior methods, scientist had to create several copies of the desired DNA sample. This DNA sample needed to be randomly broken into segments that were then put through a machine that read the chemical signals of the DNA. Thanks to the new CRISPR/nanopore method, desired genes can be selected much faster and studied more accurately. However, this new method does have its limitations. Records of the preexisting changes of the genes are lost, there is a high input requirement, the protocol is long, and the number of complete DNA reads obtained is low.
Gilpatrick, T., et al. 2020. Targeted nanopore sequencing with Cas9-guided adapter ligation. Nature Biotechnology.