CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency.
Fig: In vitro cleavage assay and OmB cell line gene targeting of IMPA1.1 using Cas9/gRNA RNPs. (a) Gene map of Oreochromis IMPA1.1 showing selected gRNA target sites and expanded view of T7 site containing BsrGI restriction site used for analysis of Cas9 cleavage. (b–e) Agarose gel images with marker sizes (base pairs or bp) labeled in red (b and c) of initial in vitro cleavage assay screen of ten target sites (T1-T10) and reference un-digested substrate amplicon (UD); (d) Follow up confirmatory in vitro cleavage assay results of the apparent most efficient cleaving gRNAs from the initial screen showing complete cleavage of substrate amplicon into expected fragments and (e) RSM analysis in which the BsrGI digested amplicon from IMPA1.1 T7 CRISPR treated cells (CR D) shows the exact same band pattern as the BsrGI digested wild-type amplicon (WT D) indicating no detectable mutation at the target site. The un-digested amplicons were included for reference (WT UD = wild-type un-digested, CR UD = CRISPR treated un-digested). The gel images in this figure have been significantly cropped to conserve space and increase focus on relevant bands. Full-length gels are presented in Supplementary Figure. Gene map and sequence images were generated using Geneious 11.0.3 (Biomatters). Image editing and assembly into complete figures was performed using Inkscape 0.92.
In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.
Hamar, J., Kültz, D. An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters.Sci Rep 11, 7854 (2021). https://doi.org/10.1038/s41598-021-87068-3