Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9-mediated genome editing events

In SDN-3 applications of CRISPR/Cas9 in mice, it was shown that DNA templates are incorporated multiple times within target sequences and cannot be detected using simple standard analysis methods (PCR, polymerase chain reaction). In the study, the authors intended to create mice containing a conditional knockout of a certain gene. For this purpose, together with the gene scissors CRISPR/Cas9, two specific guide RNAs (guides of the gene scissors to identify the target sequence) and a DNA template were introduced into the mouse embryos by direct injections. It turned out that in a large proportion of the genome-edited offspring, the DNA template was incorporated several times within the target sequence. The same effect occurred repeatedly in further experiments and appears to be a general phenomenon when using CRISPR/Cas9. Surprisingly, it was not possible to detect these multiple integrations with conventional PCR methods; for this purpose, the target area must be examined more closely with various PCR analyzes or a method called Southern blotting. It is therefore important, after genome editing, to examine the target area in the genome in detail for unintentionally incorporated DNA sequences.