Knock-in Blunt Ligation Utilizing CRISPR/Cas9

The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation ( Auer et al., 2014 ; Byrne et al., 2015 ). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks ( Jinek et al., 2012 ) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery ( Geisinger et al., 2016 ). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells. Here we present a generalized protocol for knock-in blunt ligation, using HEK293 cells as an example.