“A single-cell approach to cancer mutation discovery and CRISPR phenotypic modeling” was the title of the London Calling 2023 session by Hanlee Ji from Stanford University. Ji spoke about the use of single cell genomics and variant analysis. They spoke about how cancer mutations and clonal variation can be detected. In addition, CRISPR technologies allow for single-cell engineering. Ji noted that single-cell RNA sequencing is becoming ubiquitous. Nanopore technologies and RNA sequencing at the single-cell level allow for isoform discovery. Ji used single cell mutation analysis and adaptive sampling using a gene list. Cell barcode matching can be paired with long and short reads. Ji and team started with short reads from Illumina sequencing and merged that dataset with long-read sequencing data. They tested this system with a “proof of concept single cell study” to analyze cancers. Ji explained that they did have a “ground truth dataset” that was extensively characterized. Long reads were processed with minimap and variant calling. Epithelial and B cell metastatic cancers were studied. For cancer 1 sample, single cell transcriptomes from two sites were studied. Using adaptive sampling, 330 cancer genes were sequenced from omentum and the small bowel sites. Ji noted that their sensitivity was a lower because of the low output from the MinION system. They are transitioning to a PromethION system that will increase output and resolution. In a second cancer, transcripts from individual cells encoded the mutations they had identified. In the third cancer, a series of mutations were identified and phased. A fusion gene and BCL2 rearrangement was observed in a single cell. Using CRISPR engineering, the reverse approach was taken by Ji and team. Single cell genomics and CRISPR editing were used to introduce desired mutations and Nanopore sequencing at the single-cell level was then used to detect variant transcripts. With this approach, single-cell point mutations can be introduced in a highly multiplexed experiment. The research team functionally characterized some of the mutations. Ji ended by explaining that spatial analysis can be performed with nanopore sequencing too. Their team concluded that it is feasible to identify cancer mutations and rearrangements at the single-cell level with long-read sequencing and adaptive sampling. The integration of sequencing and CRISPR engineering at the single-cell genomics level is impressive… and will likely lead to additional approaches and technologies.
