Higher Order Multiplexing Using Digital PCR for CRISPR Gene Edit Validation

Abstract
‘Higher order multiplexing’ is the unique ability of digital PCR (dPCR) to precisely measure more targets than there are detection channels in the same reaction. In quantitative PCR, in order to measure three targets, three detection channels are required, however, in dPCR, the partitioning of the reaction into discrete partitions prior to the PCR enables three or more targets to be detected with two detection channels. This is achieved by varying the amount of primers and/or probes added to the reaction so that the end-point fluorescence is different between two targets with the same detection probe; the end-point fluorescence generates different “clusters” of partitions that can be observed on a 2D scatter plot. This talk will describe the different multiplex assay design strategies we have developed for genotyping and how precise and sensitive quantification can be achieved from counting the number of partitions in each of the clusters. The main example will illustrate our assay design for genotyping knock-in and knock-out CRISPR gene edits using a combined tandem probe binding and drop off probe design that we use to validate and determine the efficiency of desired gene edit.