Harnessing the natural diversity and in vitro evolution of Cas9 to expand the genome editing toolbox

In the past few years, the Cas9 endonuclease from the type II CRISPR-Cas bacterial antiviral defense system has revolutionized the genome editing field. Guided by an RNA molecule, Cas9 can be reprogrammed to target almost any DNA sequence: the only limitation being the short nucleotide sequence in the vicinity of the target, termed the PAM, which is characteristic for each Cas9 protein. Streptococcus pyogenes Cas9 which recognizes the NGG PAM is currently most widely used for genome manipulation. However, Cas9 orthologues and engineered Cas9 variants offer expanded genome targeting capabilities, improved specificity and biochemical properties.