Yang L, Yang JL, et al. (2014) CRISPR/Cas9-directed genome editing of cultured cells. Curr Protoc Mol Biol, 107(31):1–17.
The discovery of the CRISPR/Cas9 system is a major advancement in targeted genome editing, heralding great potential for functional research and therapeutic applications. The system uses a short guide RNA (gRNA) to direct Cas9 to genomic sequences. Cas9 cleaves the target DNA producing either a deletion, or allowing for insertion of a new donor sequence by homologous recombination.
This publication from the lab of Dr George Church at Harvard University describes a complete protocol for designing gRNAs from gBlocks® Gene Fragments
, and transferring the gRNA and Cas9 constructs into human-induced pluripotent stem cells and HEK293 cells. It also contains methods for assessing the success of the genome modification and isolating cells with the targeted changes. While the protocol is specific to 2 types of human cell lines, it should also provide useful information that is applicable to other cell culture models.