CRISPR/Cas9 Genome Editing in Embryonic Stem Cells.

Targeted mutagenesis is required to evaluate the function of DNA segments across the genome. In recent years the CRISPR/ Cas9technology has been widely used for functional genomestudies and is partially replacing classical homologous recombinationmethods in different aspects. CRISPR/ Cas9-derived tools indeed allow the production of a wide-range of engineered mutations: from point mutationsto large chromosomal rearrangementssuch as deletions, duplications and inversions. Here we present a protocol to engineer Embryonic Stem Cells(ESC) with desired mutations using transfection of custom-made CRISPR/ Cas9vectors. These methods allow the in vivo modeling of congenital mutations and the functional interrogation of DNA sequences.