Tsukamoto Tomohito, Mizuta Haruna, Sakai Eiko, Sakurai Fuminori, Mizuguchi Hiroyuki
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan; Laboratory of Functional Organoid for Drug Discovery, Center for Drug Discovery Resources Research, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan; Global Center for Medical Engineering and Informatics, Osaka University, Osaka, Japan; Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, Osaka, Japan; Center for Infectious Disease Education and Research (CiDER), Osaka University, Osaka, Japan.
J Pharm Sci. 2025 Feb;114(2):841-848. doi: 10.1016/j.xphs.2024.10.029. Epub 2024 Oct 24.
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.
使用CRISPR-Cas系统的基因组编辑技术不仅作为一种用于基因组功能分析的有前景的实验工具，而且作为一种用于遗传疾病的新型治疗方法，正吸引着广泛关注。在各种类型的Cas蛋白中，Cas12a因其独特的特性（包括低脱靶效应）有望成为一种有前景的基因编辑工具。由于Cas蛋白起源于原核生物，它们需要与适当的定位信号融合才能在真核细胞中发挥功能。到目前为止，已经开发出与核定位信号（NLS）融合的Cas12a蛋白，但核定位活性与基因组编辑效率之间的关系尚未完全阐明。在这里，利用两种Cas12a直系同源物AsCas12a和LbCas12a，以及来自不同来源的不同数量的NLS，我们发现当在培养细胞中使用腺病毒（Ad）载体表达时，核定位的改善导致基因组编辑效率提高。然而，当它们在小鼠肝脏中表达时，在腺病毒载体给药四周后，不一定需要提高核定位活性来实现最大基因组编辑效率。这些数据表明，Cas12a蛋白在体外情况下的优化NLS修饰与体内情况不同。
