ArciTect™ Cas9 Nickase is a modified version of the Cas9 recombinant protein from Streptococcus pyogenes. This version of Cas9 contains a deactivating D10A mutation, leaving only one active nuclease domain that introduces a single-strand break on the DNA strand complementary to the guide RNA (gRNA). ArciTect™ Cas9 Nickase should be combined with gRNA—e.g. ArciTect™ sgRNA (Catalog #200-0013) or a duplex composed of ArciTect™ tracrRNA (Catalog #76016) and ArciTect™ crRNA (Catalog #76010)—to form a ribonucleoprotein (RNP) complex. Typically, two gRNAs are targeted to opposite DNA strands, and the two single-strand breaks create an overhanging double-strand break of defined length. This method enables the creation of large deletions for knockout models and further reduces off-target cleavage events for gene repair or insertions via homology-directed repair. ArciTect™ Cas9 Nickase also contains a nuclear localization signal at the N-terminus, ensuring that the RNP complex translocates to the nucleus, thereby increasing the efficiency of genome editing. As the RNP complex is fully functional upon transfection, it allows for immediate activity following translocation to the nucleus. The RNP complex is degraded over 48 hours, allowing sufficient time for genome editing to occur while reducing off-target effects that can be caused by the continuous presence of the RNP complex. Using the RNP system also circumvents the laborious process of generating stable Cas9-expressing cell lines, saving time and reducing the risk of off-target effects due to leaky inducible expression systems. The S. pyogenes Cas9 uses the protospacer adjacent motif (PAM) sequence NGG (where N can be any nucleotide). The enzyme will not cleave without a genomic PAM site downstream of the target sequence.