Quantification of designer nuclease induced mutation rates: a direct comparison of different methods

Abstract

Designer nucleases are broadly applied to induce site-specific DNA double-strand breaks (DSB) in genomic DNA. These are repaired by nonhomologous end joining leading to insertions or deletions (in/dels) at the respective DNA-locus. To detect in/del mutations, the heteroduplex based T7-endonuclease I -assay is widely used. However, it only provides semi-quantitative evidence regarding the number of mutated alleles. Here we compared T7-endonuclease I- and heteroduplex mobility assays, with a quantitative polymerase chain reaction mutation detection method. A zinc finger nuclease pair specific for the human adeno-associated virus integration site 1 (AAVS1), a transcription activator-like effector nuclease pair specific for the human DMD gene, and a zinc finger nuclease- and a transcription activator-like effector nuclease pair specific for the human CCR5 gene were explored. We found that the heteroduplex mobility assays and T7-endonuclease I - assays detected mutations but the relative number of mutated cells/alleles can only be estimated. In contrast, the quantitative polymerase chain reaction based method provided quantitative results which allow calculating mutation and homologous recombination rates in different eukaryotic cell types including human peripheral blood mononuclear cells. In conclusion, our quantitative polymerase chain reaction based mutation detection method expands the array of methods for in/del mutation detection and facilitates quantification of introduced in/del mutations for a genomic locus containing a mixture of mutated and unmutated DNA.

Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B

“…Next we conducted HDR with the Tet-on-inducible single-plasmid system (P1, P2, and P3) and the two-component system (CRISPR/Cas9 plasmid and the cFIXmod PCR product) in Huh7-cFIXmut, PLC/PRF/5-cFIXmut, and Hep3B-cFIXmut cells. At 72 h post-transfection, we measured gene correction efficiencies using our previously established amplification-refractory mutation system based on qPCR analysis (ARMS-qPCR) approach 30 . The ARMS-qPCR contains two pairs of primers, the reference primer pair and the detection primer pair (Figure 4A).…”

Designer nuclease‐mediated gene correction via homology‐directed repair in an in vitro model of canine hemophilia B

“…The T7E1 assay also showed, at a gDNA level, designer nuclease efficiencies similar to those in the transfection experiments using extrachromosomal targets (Figure B). To further confirm designer nuclease activities, we used a previously reported ARMS‐qPCR approach, which represents a sensitive method for quantifying the introduced insertions and deletions (InDels) of the generated designer nucleases. As shown in Figure A, TALENs showed efficiences of 24.91% (CMV‐cFIX1) and 19.49% (CMV‐cFIX2), confirming the results of the T7E1 assay.…”

“…Taken together, this makes the screening for positive HDR events more challenging. Therefore, we modified a previously described qPCR‐based method . The ARMS‐qPCR assay is a sensitive method enabling the detection of positive HDR events even with low efficiencies.…”

“…The efficiency of the designer nucleases was also determined with the amplification‐refractory mutation system (ARMS)‐qPCR approach . The outer PCR (outPCR) using primers cFIX gDNA fwd1c and cFIX check rev was utilized for comparison of all samples (PCR is not influenced by possible InDels).…”

“…For detection of positive HDR events, a modified version of an established ARMS‐qPCR was used . For each approach, two qPCR reactions were performed.…”