An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

Abstract

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. 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.

Genome editing in East African cichlids and tilapias: state-of-the-art and future directions

“…Astatotilapia burtoni females with defective sexual behaviour/hormonal signalling cell line system establishment [12] multiple target genes tested (including impa1.1, nfat5, and nr3c1)…”

Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system

“…Half of the wells that were transfected with each myca sgRNA were then used for analyzing myca ko e ciency. This was done as previously described (Hamar and Kültz 2021). Brie y, medium was removed and cells surviving hygromycin B treatment were rinsed with Dulbecco's phosphate buffered saline (DPBS, Gibco, 14190-144).…”

“…No off-target genes were identi ed that matched any of the three top scoring myca sgRNAs suggested by CRISPOR and SYNTHEGO tools. These top three sgRNAs were cloned into an optimized tilapia sgRNA expression vector, as describe previously (Hamar and Kültz 2021). Complementary oligonucleotides (Euro ns Genomics) comprising each sgRNAs forward and reverse sequences (Table S1) were annealed to generate a ClaI restriction site at the 5' end a XbaI restriction site at the 3' end.…”

“…Chinook salmon cell lines were also used to demonstrate the functionality of a vector-based expression system (Escobar-Aguirre et al 2019), as well as to optimize lentivirus-mediated infection for e cient delivery of recombinant DNA into host cells (Gratacap, Regan, et al 2020). Our lab has recently optimized and established a vector-based CRISPR/Cas9 platform for tilapia cell lines (Hamar and Kültz 2021). This study was the rst to enable in vitro gene targeting in euryhaline tilapia cells.…”

Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system

“…Chinook salmon cell lines were also used to demonstrate the functionality of a vector-based expression system 30 , as well as to optimize lentivirus-mediated infection for efficient delivery of recombinant DNA into host cells 31 . Our lab has recently optimized and established a vector-based CRISPR/Cas9 platform for tilapia cell lines 32 . This study was the first to enable in vitro gene targeting in euryhaline tilapia cells.…”