Search-and-replace genome editing without double-strand breaks or donor DNA

Prime editing, a new variant of the CRISPR/Cas toolbox, enables the introduction of deletions, insertions and all 12 options to convert one base enzymatically into another at target sequence(s) in the genome of target organisms. In prime editing, a single strand of the DNA is cut to reduce the occurrence of off-target effects. In addition, the CRISPR/Cas system has been modified in a way that the guide RNA (gRNA) for target site recognition has a second function: it also serves as a repair template to introduce an intended change at the target sequence. Another enzyme, the so-called reverse transcriptase, is linked to the gene scissors. A reverse transcriptase can be seen as a translation program that converts the gRNA into DNA. This piece of DNA then serves as a DNA template to repair the single-strand break at the DNA target site and is copied into that region of the genome. The two DNA strands at the target site are not compatible to each other anymore, thus the gene scissors cut the unchanged DNA single strand. The DNA template that has already been copied into the modified DNA single strand is then copied in the second cut site.