{"title":"Evaluation of the correlation between nuclear localization levels and genome editing efficiencies of Cas12a fused with nuclear localization signals.","authors":"Tomohito Tsukamoto, Haruna Mizuta, Eiko Sakai, Fuminori Sakurai, Hiroyuki Mizugchi","doi":"10.1016/j.xphs.2024.10.029","DOIUrl":null,"url":null,"abstract":"<p><p>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 in vitro situations differed from that in in vivo.</p>","PeriodicalId":16741,"journal":{"name":"Journal of pharmaceutical sciences","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of pharmaceutical sciences","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1016/j.xphs.2024.10.029","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"CHEMISTRY, MEDICINAL","Score":null,"Total":0}
引用次数: 0
Abstract
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 in vitro situations differed from that in in vivo.
使用CRISPR-Cas系统的基因组编辑技术不仅是分析基因组功能的一种有前途的实验工具,而且也是治疗遗传疾病的一种新方法,因此备受关注。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,有望成为一种前景广阔的基因编辑工具。由于 Cas 蛋白来源于原核细胞,因此需要融合适当的定位信号才能在真核细胞中发挥作用。目前已开发出融合了核定位信号(NLS)的 Cas12a 蛋白,但其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,我们利用两种Cas12a直向同源物AsCas12a和LbCas12a,以及来自不同来源的不同数量的NLS,发现当使用腺病毒(Ad)载体在培养细胞中表达时,改进的核定位导致基因组编辑效率的提高。然而,当它们在小鼠肝脏中表达时,核定位活性的改善并不一定要求在使用腺病毒载体四周后达到最高的基因组编辑效率。这些数据表明,Cas12a蛋白在体外的优化NLS修饰与体内不同。
期刊介绍:
The Journal of Pharmaceutical Sciences will publish original research papers, original research notes, invited topical reviews (including Minireviews), and editorial commentary and news. The area of focus shall be concepts in basic pharmaceutical science and such topics as chemical processing of pharmaceuticals, including crystallization, lyophilization, chemical stability of drugs, pharmacokinetics, biopharmaceutics, pharmacodynamics, pro-drug developments, metabolic disposition of bioactive agents, dosage form design, protein-peptide chemistry and biotechnology specifically as these relate to pharmaceutical technology, and targeted drug delivery.