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Optimization of Cas9 nuclear localization in a tilapia ( Oreochromis mossambicus ) cell line
Author(s) -
Villapando Angelika Marie,
Hamar Jens Carlton,
Kültz Dietmar
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06452
Subject(s) - oreochromis mossambicus , tilapia , line (geometry) , fishery , chemistry , biology , fish <actinopterygii> , mathematics , geometry
CRISPR gene targeting studies in cell lines require reliable expression of Cas9 along with appropriate target‐specific guide RNAs. Effective CRISPR/Cas9 gene editing requires Cas9 localization in the nucleus. Previous attempts to implement CRISPR/Cas9 gene editing in a tilapia ( Oreochromis mossambicus ) cell line (OmB cells) were unsuccessful. A possible contribution to these results could be inefficient nuclear localization of Cas9. Conventionally, the mammalian SV40 nuclear localization signal (SV40 NLS) is used to localize proteins to the nucleus. Our previous studies have shown that the SV40 promoter is poorly recognized in OmB cells. Therefore, we hypothesized that the SV40 NLS will also be poorly recognized in OmB cells. To test this hypothesis we fused the SV40 NLS to the C‐terminus of GFP (green fluorescent protein), transfected it into the tilapia OmB cell line, and determined the efficiency of nuclear GFP localization using fluorescence imaging (Leica Dmi8). Our imaging results show that the SV40 NLS moderately induced nuclear GFP localization. An incomplete but significant increase in nuclear GFP localization relative to the control (no NLS, just GFP) was observed using Leica image analysis software (LASX 2.0, Leica). Nevertheless, a large amount of GFP still remained in the cytosol when using SV40 NLS. Current work is comparing the efficiency of SV40 NLS mediated nuclear localization of GFP in fish cell lines to that mediated by an endogenous tilapia NLS. We have cloned an endogenous tilapia NLS and are currently testing whether this endogenous NLS improves nuclear localization of ectopic GFP in tilapia OmB cells. Optimization of nuclear localization of Cas9 will aid future gene editing studies in the OmB cell line and in cells from related fish species. Support or Funding Information This study was funded by a grant from NSF (IOS‐1656371).