CRISPR/Cas9 knockins

In addition to creating indels or knockouts, scientists can encourage a precise form of repair (homology-directed repair; HDR) by providing a DNA sequence that the cell can use as a repair template to insert (knock in) a matching DNA sequence into the break. Example applications include modification of a promoter sequence or gene, insertion of an exogenous reporter (e.g., a fluorescent protein), or creation of a clincally relevant SNP for a disease model. It should be noted that knockin efficiency is usually lower than knockout efficiency. Moreover, knockin at one copy of the gene/sequence is usually accompanied by a knockout/indel at the second copy; both copies are cut by the Cas9/sgRNA complex but one is likely repaired imprecisely using the non-homologous end joining (NHEJ) pathway, creating an indel.

The links below provide general information, tech notes, and tools used to create or study gene knockins. Although many template formats (plasmids, PCR products, viruses, transposons, etc.) can be used for HDR, the use of single-stranded DNA (ssDNA) templates is increasing rapidly due to the growing understanding that ssDNA integration is far more specific and less toxic than double-stranded DNA (dsDNA) templates.