Gene Editing Event Detection
This presentation outlines three assays developed using digital PCR (dPCR). The first assay was developed to detect CRISPR Cas9 gene editing events in vivo founder mice. One problem is the genetic variability introduced by gene editing mouse lines. This variability among founder mice requires efficient genotype identification to expedite the management of the mouse colony. Falabella’s team set about developing a quick and cost-efficient assay that can define the genetic variability that arises from CRISPR Cas9 gene editing.
Starting with two TaqMan drop-off probe assays, the team improved the sensitivity by switching from qPCR to dPCR; they also experimented with locked nucleic acid (LNA) probes to further enhance detection sensitivity. The drop-off assay combined with dPCR provided a rapid approach for the initial identification and selection of homology-directed repair (HDR) edits in founder mice.
mtDNA Copy Number Analysis
The second assay aimed to quantify mitochondrial DNA (mtDNA) in patients with mtDNA depletion syndromes. These syndromes usually affect the heart, brain, and skeletal muscle tissues. Unfortunately, these diseases usually cause mortality in the second or third years of life. mtDNA depletion has a heterogeneous phenotype which makes it very difficult to establish a molecular diagnosis. Furthermore, biopsy is difficult to obtain in newborn patients.
The copy number of mtDNA is particularly relevant for primary mitochondrial disease. Therefore, the team developed a straightforward single plex ddPCR assay to measure the mtDNA copy number as a diagnostic service. Analysis unequivocally showed that supports pathogenicity of a genetic variant in an SSBP1 patient.
mtDNA Deletion
Finally, Falabella outlined the ongoing development of a novel ddPCR assay to quantify mtDNA deletions as tool for understanding the mechanism of disease and to establish molecular diagnosis.
