CRISPR-Nanopore Sequencing Combo Could Revolutionize Cancer Gene Identification
The speed and accuracy with which current sequencing platforms can produce results are quite staggering compared to methodologies that were being utilized less than ten years ago. Yet, when it comes to making clinical decisions with respect to cancerous tumors, sequencing assays can almost never be fast enough. Thankfully though, investigators at the Johns Hopkins School of Medicine believe they may have found a solution that could improve the sequencing process for many cancer patients. The scientists report that they successfully used the CRISPR-Cas9 gene-editing system to make cuts in DNA around lengthy tumor genes, which can be used to collect sequence information, and coupled it to nanopore sequencing platforms.
"Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection," the authors wrote. "Current methods are limited by the loss of native modifications, short read length, high input requirements, low yield or long protocols."
Findings from the new study were published recently in Nature Methods through an article entitled "Targeted nanopore sequencing with Cas9-guided adapter ligation." 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.
"For tumor sequencing in cancer patients, you don't necessarily need to sequence the whole cancer genome," explained senior study investigator Winston Timp, PhD, assistant professor of biomedical engineering, 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 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.
In the current study, the Hopkins team was 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.
The researchers described a process called "nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adapters for nanopore sequencing. We showed that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants, structural variations and CpG methylation. We apply nCATS to four cell lines, to a cell-line-derived xenograft, and to normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675× using a MinION flow cell and 34× using the smaller Flongle flow cell. The nCATS sequencing requires only ~3 μg of genomic DNA and can target a large number of loci in a single reaction."
Among 10 breast cancer genes the Hopkins team focused 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 tumor 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 tumor 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 gentler with DNA from human tissue samples because it's been frozen and thawed several times," Timp remarked.
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.
"This gene is really long, and we were able to collect sequencing reads which went all the way through this large and complex region," noted lead study investigator Timothy Gilpatrick, an MD/PhD student in Timp's laboratory.
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. While they were excited by their findings, the researchers plan to continue refining the CRISPR/nanopore sequencing technique and testing its capabilities in other tumor types.
"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," Timp concluded.
Ubigene Biosciences is co-founded by biological academics and elites from China, the United States, and France. We are located in Guangzhou Science City, which serves as a global center for high technology and innovation. Ubigene Biosciences has 1000㎡ office areas and laboratories, involving genome editing, cell biology technology, and zebrafish research. We provide products and services for plasmids, viruses, cells, and zebrafish. We aim to provide customers with better gene-editing tools for cell or animal research.
We developed CRISPR-U™ and CRISPR-B™ (based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, CRISPR-U™ and CRISPR-B™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo.
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