our study uncovered serious pitfalls exemplified in 10 separate KI procedures during the construction of six cKO mouse models that need to be taken into account. All gene-targeting protocols were performed by direct injection of CRISPR-Cas9 components together with donor DNA template into fertilized oocytes. Eight KIs were performed with relatively long donor DNA fragments (~700 to 1650 nt). Seven procedures used ssDNA and three dsDNA templates (Table 1). Three KI attempts with ssDNA and one with dsDNA templates did not yield the intended single copy integration of donor template (Table 1).
Efficiencies of donor DNA integration were variable and correlated with template size; in general, longer templates integrated less efficiently (Table 1). We noticed that most edited mice obtained from CRISPR-Cas9–modified zygotes (F0 generation) exhibited mosaic genotypes, harboring subpopulations of cells derived from different DNA integration events, and contained diverse copy numbers in the targeted loci. Our data suggest that PCR amplification of short genomic flanking regions in conjunction with inserted donor DNA is the most efficient and reliable approach for the identification of F0 mice with correctly targeted loci. Positive PCR results on both flanks indicated that a certain subpopulation of cells contains HDR-integrated DNA template (Fig. 1, C and D). However, longer PCR products representing subpopulations of cells with target DNA integrated via HDR-NHEJ or NHEJ-NHEJ are difficult to amplify. Nevertheless, in some cases, most probably depending on the degree of mosaicism and PCR primer locations, these arrangements could be detected as well (Fig. 1D, numbers 10 and 18).
When the selected F0 founders were crossed with wild-type mice for F1 offspring production, we often detected animals harboring multiple head-to-tail integrations of the donor template at the targeted loci (Fig. 3). We observed template multiplication irrespective of size, nucleotide composition, or the utilization of dsDNA or ssDNA (Table 1). A commonly applied PCR verification method in heterozygotic animals using template-specific primers in most cases erroneously identified those as single copy integration events. Moreover, in cases of multiple-copy HDR-HDR–based integrations of donor DNA, it proved impossible to correctly identify the desired single copy mice by amplification with primers set in the genomic flanking regions followed by PCR product sequencing.