The RNA transcript can at least in principle act as a repair template, this is referred to as RNA-templated DNA repair [45].
When injecting the editing components, the authors also included a nucleotide sequence containing wild-type MYBPC3 that could be used as a repair template by the editing machinery.
Their results indicate that the wild-type maternal copy of MYBPC3 probably provided the repair template, rather than the introduced copy.
Mos1 excision generates a DNA double-strand break, which is repaired by homologous recombination using a transgenic repair template.
The authors designed the repair template to encode the same amino acids as the wild-type maternal copy of the gene, but used some different nucleotides so that they could distinguish between repair using either the maternal copy of MYBPC3 or the introduced repair template.
Conventional wisdom among CRISPR-Cas9 researchers has held that the tool randomly generates insertions and deletions in a gene unless researchers include a so-called repair template.
This is bound by replication protein A (RPA), and, during mitotic recombination, a Rad51 nucleofilament is formed promoting strand invasion of the sister chromatid or homologous chromosome, which is used as a repair template before second end capture3.
While NHEJ is active throughout interphase, HRR is restricted to the S and G2 phases of the cell-cycle, where a homologous sister chromatid is available as repair template.
The other more difficult option allows genes to be corrected or new genes to be inserted as the cell copies a DNA repair template that is delivered alongside the cutting machinery.
Imprecise repair of the break can yield mutations, while homologous recombination with a repair template can be used to effect specific changes to the genome.
Genome editing with engineered nucleases has recently emerged as an approach to correct genetic mutations by enhancing homologous recombination with a DNA repair template.
