Abstract
We describe a method for measuring genome editing efficiency from in silico analysis of high-resolution melt curve data. The melt curve data derived from amplicons of genome-edited or unmodified target sites were processed to remove the background fluorescent signal emanating from free fluorophore and then corrected for temperature-dependent quenching of fluorescence of double-stranded DNA-bound fluorophore. Corrected data were normalized and numerically differentiated to obtain the first derivatives of the melt curves. These were then mathematically modeled as a sum or superposition of minimal number of Gaussian components. Using Gaussian parameters determined by modeling of melt curve derivatives of unedited samples, we were able to model melt curve derivatives from genetically altered target sites where the mutant population could be accommodated using an additional Gaussian component. From this, the proportion contributed by the mutant component in the target region amplicon could be accurately determined. Mutant component computations compared well with the mutant frequency determination from next generation sequencing data. The results were also consistent with our earlier studies that used difference curve areas from high-resolution melt curves for determining the efficiency of genome-editing reagents. The advantage of the described method is that it does not require calibration curves to estimate proportion of mutants in amplicons of genome-edited target sites.
Significance Statement Genome editing has been revolutionized by the engineering of molecular scissors that cut DNA at a predetermined location on the chromosome. When these molecular scissors are expressed within cells, these scissors cut the genomic DNA at the designated target site, and the cells respond by repairing the cut sites. This repair process frequently introduces mutations at the target cut site. The more efficient the molecular scissors, the more number of cells in a treated culture dish exhibit these mutations at the cut site. Investigators therefore design several molecular scissors targeting the same region on the chromosome to identify the best ones. We describe a new recipe to measure scissors’ efficiency in target site cutting.
Footnotes
MZ: michail.zaboikin{at}health.slu.edu, CF: carl.freter{at}health.slu.edu