Evaluation of the correlation between nuclear localization levels and genome editing efficiencies of Cas12a fused with nuclear localization signals

• Published in: Journal of pharmaceutical sciences 2024-10
• Main Authors: Tsukamoto, Tomohito, Mizuta, Haruna, Sakai, Eiko, Sakurai, Fuminori, Mizuguchi, Hiroyuki
• Format: Article
• Language: English
• Subjects: Adenovirus vector; Cas12a; Gene therapy; Genome editing; Nuclear localization signal
• ISSN: 0022-3549; 1520-6017; 1520-6017
• DOI: 10.1016/j.xphs.2024.10.029

•Nuclear localization of Cas12a was significantly enhanced by addition of multiple NLSs in cultured cells.•The nuclear localization levels of Cas12a were not always correlated with the genome editing efficiencies in cultured cells.•The nuclear localization of Cas12a proteins was significantly improved in the tissues as the numbers of NLSs were increased.•The effects of NLSs on in vitro genome editing efficiencies of Cas12a did not always correlate with those on in vivo. Genome editing technology using the CRISPR-Cas system is attracting much attention not only as a promising experimental tool for analysis of genome functions, but also as a novel therapeutic approach for genetic disorders. Among the various types of Cas proteins, Cas12a is expected to be a promising gene editing tool due to its unique properties, including low off-target effects. As Cas proteins are of prokaryotic origin, they need to be fused with appropriate localization signals to perform their function in eukaryotic cells. Cas12a proteins fused with a nuclear localization signal (NLS) have been developed so far, but the relation between the nuclear localization activity and the genome editing efficiency has not been fully elucidated. Here, utilizing two Cas12a orthologs, AsCas12a and LbCas12a, with various number of NLSs derived from various origins, we revealed that the improved nuclear localization resulted in increased genome editing efficiencies when expressed using adenovirus (Ad) vector in cultured cells. However, when they were expressed in mouse liver, the improvement of the nuclear localization activity was not necessarily required to achieve the maximum genome editing efficiency four weeks after Ad vector administration. These data indicated that the optimized NLS modification of Cas12a proteins in vitro situations differed from that in vivo.