A new sequencing approach has been developed by scientists at KAUST that can be used to detect rare mutations in large groups of cells. The sequencing techniques currently being used lack that level of sensitivity and are unable to detect rare gene mutations in large groups of cells. The new approach – named targeted individual DNA molecular sequencing – IDMseq – allows a single mutation to be detected in a group of 10,000 cells.
The new technique identifies the rare mutations and attaches a barcode to the cells, each of which is unique, then large numbers of copies of each molecule are made using a polymerase chain reaction (PCR), with each copy having the same barcode as the original.
Variant analysis with unique molecular identifier for long-read technology (VAULT), a bioinformatics toolkit, is used to decode the barcodes and sort similar molecules into bins, with each bin representing one of the original DNA molecules. The VAULT system is then used to detect mutations in each of the bins, allowing scientists to determine the types of mutation and the frequency. This approach allows the scientists to detect even small single-base mutations, as well as large insertions and deletions of sections of DNA. Identifying rare mutations is important in early cancer detection and would help to identify cancerous changes quickly, at a time when treatment is likely to be the most effective. The system can also be used to check for off-target effects in CRISPR edited cells.
The KAUST scientists demonstrated their sequencing technique by identifying the number and frequency of gene edits performed by the CRISPR-Cas9 gene editing tool in human embryonic stem cells. When the CRISPR-edited cells were mixed with wild-type cells, the system was able to detect the mutation at ratios of 1:100, 1:1,000, and 1:10,000 and correctly reported the frequency of the mutation. The system can only currently work on one strand of DNA, but the researchers hope to develop a system that will work on double stranded DNA.
“Several recent studies have reported that Cas9 introduces unexpected, large DNA deletions around the edited genes, leading to safety concerns,” said Ph.D. student Chongwei Bi. “These deletions are difficult to detect and quantitate using current DNA sequencing strategies. But our approach, in combination with various sequencing platforms, can analyze these large DNA mutations with high accuracy and sensitivity.”
Using the new sequencing technique the researchers determined that in a sample of CRISPR-Cas9 edited cells, large deletions accounted for around 2.8-5.4% of outcomes, but there was also a 300% increase in single-base DNA variants in the edited region of DNA.
“This shows that there is a lot that we need to learn about CRISPR/Cas9 before it can be safely used in the clinic,” said co-author Yanyi Huang, PhD, of Peking University.
You can read more about the sequencing technique in the paper – Long-read individual-molecule sequencing reveals CRISPR-induced genetic heterogeneity in human ESCs – which was recently published in the journal Genome Biology. DOI: 10.1186/s13059-020-02143-8