Massively parallel phenotyping of variant impact in cancer with Perturb-seq reveals a shift in the spectrum of cell states induced by somatic mutations

Abstract

Genome sequencing studies have identified millions of somatic variants in cancer, but their phenotypic impact remains challenging to predict. Current experimental approaches to distinguish between functionally impactful and neutral variants require customized phenotypic assays that often report on average effects, and are not easily scaled. Here, we develop a generalizable, high-dimensional, and scalable approach to functionally assess variant impact in single cells by pooled Perturb-seq. Specifically, we assessed the impact of 200 TP53 and KRAS variants in >300,000 single lung cancer cells, and used the profiles to categorize variants into phenotypic subsets to distinguish gain-of-function, loss-of-function and dominant negative variants, which we validated by comparison to orthogonal assays. Surprisingly, KRAS variants did not merely fit into discrete functional categories, but rather spanned a continuum of gain-of-function phenotypes driven by quantitative shifts in cell composition at the single cell level. We further discovered novel gain-of-function KRAS variants whose impact could not have been predicted solely by their occurrence in patient samples. Our work provides a scalable, gene-agnostic method for coding variant impact phenotyping, which can be applied in cancer and other diseases driven by somatic or germline coding mutations.

Identifying disease-critical cell types and cellular processes across the human body by integration of single-cell profiles and human genetics

“…Looking forward, the gene program-disease links identified by our analyses can be used to guide downstream studies, including designing systematic perturbation experiments (161,162)…”

“…Looking forward, the gene program-disease links identified by our analyses can be used to guide downstream studies, including designing systematic perturbation experiments( 161, 162 ) in cell and animal models( 163 ) for functional follow up. Additionally, the continued growth of the Human Cell Atlas( 164 ) as a reference as well as cell atlases across many diseases, will help to more specifically map the cellular mechanisms underlying a disease across varying contexts.…”

Variant Library Annotation Tool (VaLiAnT): an oligonucleotide library design and annotation tool for Saturation Genome Editing and other Deep Mutational Scanning experiments

“…VaLiAnT has been designed to aid in the design and generation of oligonucleotide libraries for SGE experiments. However, VaLiAnT has a wide range of possible uses including, but not limited to: mutational consequence annotation of coding-sequence variation, conversion of VCF annotation files to oligonucleotide sequences and the generation of libraries for exogenous or orthogonal assays (as opposed to genome editing), that use cDNA cassettes and/or transcriptional readouts (Jones et al, 2020; Ursu et al, 2020). The reverse-complement option in VaLiAnT permits the generation of variant sequences for strand-specific applications.…”

Linking genome variants to disease: scalable approaches to test the functional impact of human mutations

“…An alternative approach to layering multiple assays would be to use a single assay capable of capturing different functional classes of variants. Of note, one group has recently demonstrated using single-cell RNA-sequencing to interrogate patient variants observed in the oncogenes NRAS and MYC ( 102 ). Coupling expressed barcodes to each variant allowed cells to be genotyped and transcriptionally profiled, revealing distinct pathways activated by specific mutations.…”