The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is the latest RNA-guided, endonuclease tool in genome editing which allows for very specific genomic disruption and replacement. One concern with the current CRISPR Cas9 technology is the potential off-target effects of the Cas9 nuclease. ;To counteract off-target mutagenic effects of this system, the Cas9 Nickase D10A was developed with a D10A mutation in its RuvC1 nuclease domain. Unlike the Cas9 nuclease, this mutant form generates a single stranded nick instead of a double-strand break (DSB). Because a single DNA nick is quickly repaired with high fidelity by the cellular machinery, the system requires two closely juxtaposed nicks in order to trigger the same genomic disruption as the Cas9 nuclease. This effectively boosts the recognition sequence to 40 instead of 20 nucleotides, and, as a result, off-target effects become highly unlikely. Thus, the double-nickase CRISPR system offers unparalleled specificity to satisfy even the most stringent of experimental requirements. ;The Cas9 from the bacteria Streptococcus pyogenes, abbreviated spCas9, is the most commonly used Cas9 variant. ;The fusion of Cas9 to GFP allows for visual confirmation of transfection as well as subsequent verification of Cas9 clearance from the cells. Cas9 D10A Nickase-GFP can also be used for FACS applications and screening. Cas9 D10A Nickase -GFP NLS contains a SV40 T antigen nuclear localization sequence (NLS) on the C-terminus of the protein.