Direct measurement of engineered cancer mutations and their transcriptional phenotypes in single cells

Genome sequencing studies have identified numerous cancer mutations across a wide spectrum of tumor types, but determining the phenotypic consequence of these mutations remains a challenge. Here, we developed a high-throughput, multiplexed single-cell technology called TISCC-seq to engineer predesignated mutations in cells using CRISPR base editors, directly delineate their genotype among individual cells and determine each mutation's transcriptional phenotype. Long-read sequencing of the target gene's transcript identifies the engineered mutations, and the transcriptome profile from the same set of cells is simultaneously analyzed by short-read sequencing. Through integration, we determine the mutations' genotype and expression phenotype at single-cell resolution. Using cell lines, we engineer and evaluate the impact of >100 TP53 mutations on gene expression. Based on the single-cell gene expression, we classify the mutations as having a functionally significant phenotype.

Automated summary

This study developed a high-throughput, multiplexed single-cell technology called TISCC-seq to engineer mutations in cells using CRISPR base editors and determine their genotype and transcriptional phenotype. Through engineering and evaluating TP53 mutations, the study classified these mutations based on their single-cell gene expression.