Enhanced green fluorescent protein (eGFP)-tagged Cas9 nuclease for the generation of double-strand breaks in CRISPR-Cas9 genome editing.

ArciTect™ Cas9-eGFP Nuclease is a fusion protein consisting of enhanced green fluorescent protein (eGFP) and the wild-type Cas9 recombinant protein from Streptococcus pyogenes. ArciTect™ Cas9-eGFP Nuclease contains a C-terminal-linked eGFP molecule. ArciTect™ Cas9-eGFP Nuclease requires association with a guide RNA - e.g. ArciTect™ sgRNA or a duplex composed of ArciTect™ tracrRNA and ArciTect™ crRNA - to form a ribonucleoprotein (RNP) complex. This RNP complex creates double-strand breaks at site-specific locations in the genome. ArciTect™ Cas9-eGFP Nuclease also contains a nuclear localisation 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.

This RNP complex creates double-strand breaks at site-specific locations in the genome. ArciTect™ Cas9-eGFP Nuclease also contains a nuclear localisation 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.