Validation of a Novel Double Control Quantitative Copy Number PCR Method to Quantify Off-Target Transgene Integration after CRISPR-Induced DNA Modification

Abstract

In order to improve a recently established cell-based assay to assess the potency of botulinum neurotoxin, neuroblastoma-derived SiMa cells and induced pluripotent stem-cells (iPSC) were modified to incorporate the coding sequence of a reporter luciferase into a genetic safe harbor utilizing CRISPR/Cas9. A novel method, the double-control quantitative copy number PCR (dc-qcnPCR), was developed to detect off-target integrations of donor DNA. The donor DNA insertion success rate and targeted insertion success rate were analyzed in clones of each cell type. The dc-qcnPCR reliably quantified the copy number in both cell lines. The probability of incorrect donor DNA integration was significantly increased in SiMa cells in comparison to the iPSCs. This can possibly be explained by the lower bundled relative gene expression of a number of double-strand repair genes (BRCA1, DNA2, EXO1, MCPH1, MRE11, and RAD51) in SiMa clones than in iPSC clones. The dc-qcnPCR offers an efficient and cost-effective method to detect off-target CRISPR/Cas9-induced donor DNA integrations.

Determining On‐Target, Off‐Target, and Copy Number Status of Transgenic Events After CRISPR/Cas9 Targeted AAVS1 Safe‐Harbor Modification of iPSCs Using Double‐Control Quantitative Copy Number PCR

“…A limitation of this tool is that although it can be exploited to detect the specific target modification in the cells, as in the example detailed here involving the insertion of the coding sequence of Gaussia luciferase into the AAVS1 safe‐harbor locus, it cannot detect off‐target double‐strand breaks that may have resulted in point mutations rather than integration of the donor DNA. Nonetheless, it has proven to be a relevant and useful tool for detecting off‐target integrations, which, depending on cell type, have been shown to still occur frequently (Schjeide et al., 2022). The greatest benefit of this workflow is that each protocol takes advantage of techniques and devices that are commonly found in most standard molecular biology laboratories, or at least neighboring laboratories.…”

“…Therefore, the same instrument should also be capable of detecting small variations in gDNA copy number. Based on this hypothesis, we validated the sensitivity of the qPCR and developed the autosomal/ChrX double‐control quantitative copy number PCR (dc‐qcnPCR) to detect one, two, or more copies of a sequence of interest (Schjeide, Schenke, Seeger, & Püschel, 2022). The insert‐confirmation PCR is an essential partner to the dc‐qcnPCR.…”

“…The Southern blot is a standard method designed to detect specific sequences in digested gDNA separated by gel electrophoresis (Southern, 1975). We found, however, that when using non‐isotope‐based strategies, the method was not sensitive enough to detect single copies of functionally verified Gaussia luciferase coding DNA (Schjeide et al., 2022). More recent advances to assess off‐target modifications involve detection of potential Cas off‐target inserts followed up by next‐generation sequencing: for example, genome‐wide, unbiased identification of DSBs enabled by sequencing (GUIDE‐seq; Tsai et al., 2015); discovery of in situ Cas off‐targets and verification by sequencing (DISCOVER‐seq; Wienert et al., 2019); circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE‐seq; Tsai et al., 2017); and linear amplification‐mediated high‐throughput genome‐wide translocation sequencing (LAM‐HGTGTS; Frock et al., 2014).…”