CRISPR/Cas9 technology is one of the most popular methods used for genome editing. It introduces both Cas9 endonuclease and a guide RNA into the cells of interest. The guide RNA is designed to direct the Cas9 endonuclease to a particular site in the genome where it produces a double-strand break (DSB). There are generally two ways to repair the double-stranded break: non-homologous end-joining (NHEJ) or homologous-directed repair (HDR). NHEJ is the main form of repair in mammalian cells. As it is error-prone, repair via the NHEJ pathway allows for insertion, deletion, and loss-of-function mutations which probably result in frameshifts and affect protein expression. In addition to knockout mutations, a template DNA can be introduced to direct HDR and create mutations in the gene of interest. HDR faithfully copies the template sequence to the cut target site.
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