Project summary Single cell “multi-omics” represents a promising set of emerging tools to establish crucial links between transcriptional programs and their underlying drivers such as mutations and the activity of signaling pathways. These tools have broad application to understand mechanisms of disease, tissue development and regeneration. The methods involve the simultaneous analysis of the transcriptome of each cell with other measurements such as DNA sequence, epigenetics and protein composition. The leading high-throughput paradigms for single cell genomics have technical limitations that severely impact their use in multiplexing. Only a few genome-wide assays have now been multiplexed at high cell throughput, and there remains a technical challenge in maintaining good sensitivity upon multiplexing. Some joint measurements remain undemonstrated and may be impossible to implement with existing methods. We develop here a novel technology for multi-step biochemical analysis of single cells using sub-nanoliter semi-permeable compartments (or “capsules”). Capsules should allow multiple complex measurements on single cells not currently possible, and should improve the quality of data from existing single cell multi-omic efforts because they overcome the limitations of existing platforms. Cells are captured in capsules in a process that is cheap and fast, where they may be lysed and barcoded for sequencing. Unlike widely-used methods for single cell genomics that capture cells in droplets, the capsules are not isolated by oil: instead they exchange molecules with their environment, which enables multiple reaction buffer exchanges while processing single cell lysates. Nucleic acids are trapped in capsules without cross-linking. Capsules can also be sorted to allow screening and enrichment of cell lysates for analysis. This R21 proposal will bring capsules to practice in single cell multi-omics. It is rooted in preliminary data where we demonstrate the defining technical requirements of capsules. If successful, this work will establish a powerful new technology for single cell multi-modal assays that overcomes limitations of existing methods. In doing so we will implement a multi-omic assay with application to screening and cancer phenotyping, and we will set the stage for implementing additional high quality multi-omic assays.

Project Narrative This proposal develops a novel technological platform for carrying out multi-faceted measurements on large numbers of single cells. The methods developed will have broad applications to understanding the events that occur in diseases such as cancer and fibrosis, and for regenerative medicine. Within this grant, we will move from new established technical capabilities, to a first set of transformative new methods ready for use.