High Throughput Single-cell Assessment of Genome Integrity and Toxicity Events Associated With Edited Cells

Despite advancements in precise gene editing technologies such as CRISPR-Cas9, TALENs, and ZFNs, the fundamental editing process yields heterogeneous populations where some cells may have undesired outcomes that bear the risk of genome toxicity. Notably, these adverse outcomes include the introduction of structural variants, copy number alterations, or chromosomal translocations. Therefore, the development of efficacious gene therapies hinges on the ability to accurately measure and understand these events. Furthermore, since “cells” are the functional units of gene editing products, it is prudent to measure the co-occurrences of editing results and potential genotoxicity events in a single-cell context.

Here, we demonstrate a microfluidics and multiplex PCR based single-cell technology that, in once assay, simultaneously measures the co-occurrence and zygosity of on-target edit, off-target edits, translocations between predicted edit sites, as well as the genomic CNV landscape (including focal CNV) in over thousands of cells in parallel. This single-cell technology offers a comprehensive view of the heterogeneous editing profile of gene edited products for a proper and fast evaluation of editing outcome and potential malignant events.