Genetic Engineering of Human Keratinocytes Using CRISPR/Cas9 Ribonucleoprotein Complexes or Modified Cas9-Encoding mRNAs.

CRISPR/Cas9 is a straightforward genome-editing technique that is implemented across disciplines and research areas. However, in keratinocytes, CRISPR/Cas9 can be particularly difficult due to variable genome-editing efficiency, reduced cell viability, and difficulties during (sub)cloning of gene-edited keratinocyte populations. Here, we provide a step-by-step detailed protocol for the genetic manipulation of human (primary) keratinocytes, including widely accepted procedures for the analysis of CRISPR/Cas9 efficiency, (sub)cloning procedures to select heterozygous or homozygous keratinocytes, and off-target genome-editing analysis.