Publisher: Elsevier Country of Publication: United States NLM ID: 2985195R Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1520-6017 (Electronic) Linking ISSN: 00223549 NLM ISO Abbreviation: J Pharm Sci Subsets: MEDLINE

Gene Editing*/methods , Nuclear Localization Signals*/genetics , Nuclear Localization Signals*/metabolism , CRISPR-Cas Systems*/genetics , CRISPR-Associated Proteins*/genetics , CRISPR-Associated Proteins*/metabolism , Cell Nucleus*/metabolism , Cell Nucleus*/genetics , Endodeoxyribonucleases*/genetics , Endodeoxyribonucleases*/metabolism, Animals ; Mice ; Humans ; Genetic Vectors/genetics ; Adenoviridae/genetics ; HEK293 Cells ; Liver/metabolism ; Bacterial Proteins

Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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.
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