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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.
Highlights
Genome editing at predetermined loci has been greatly facilitated by new technologies based on RNA-guided endonucleases (RGENs)[1,2,3] or transcription-activator like effector nucleases (TALENs) [4,5,6]
The dimeric guide RNA-dCas9-FokI system consists of pSQT1313 and pSQT1601 plasmids. pSQT1313 is used for expression of dual guide RNAs that target genomic DNA sequences on opposite strands and spaced approximately 16 bases apart. pSQT1601 encodes dCas9-FokI fusion protein to effect double-stranded breaks (DSBs) and Csy4 RNase to process the dgRNA expressed by pSQT1313
The genomic DNA (gDNA), isolated from unselected or selected populations of transfected cells, were amplified and high-resolution melt curve data were obtained as described in Materials and Methods
Summary
Genome editing at predetermined loci has been greatly facilitated by new technologies based on RNA-guided endonucleases (RGENs)[1,2,3] or transcription-activator like effector nucleases (TALENs) [4,5,6]. The pre-melt region of a melting curve identified from plots of melt curves of unmodified or mock-transfected cells was used to determine the efficiency of detecting dsDNA-bound fluorophore at different temperatures. This region of BcRFU(x) of mock-transfected cells was plotted separately and subjected to least squares curve fitting (Eq 3). The resulting values, representing predicted RFU of unmelted DNA at the different temperatures, were normalized to the starting temperature (Tlow or 71 ̊C) to obtain the efficiency of detection of dsDNA-bound fluorophore at each measured temperature point (Eq 4). First derivatives of nFcRFU, obtained by numerical differentiation (Eq 7), were used for subsequent curve fitting analysis
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