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

Jens Hamar,Dietmar Kültz

doi:10.1038/s41598-021-87068-3

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.

Highlights

Use of fish in physiology studies is widespread with purposes ranging from production enhancement of economically important species to models of basic vertebrate biology
Targeted genetic manipulation of cell cultures has been an effective tool in deciphering specific functions of cellular components, which is enabled by applying CRISPR/Cas[9] gene editing systems to cell culture m odels[8,9]
Transfection of Cas9/gRNA ribonucleoprotein complexes (RNPs) using these targets was performed on wild-type Oreochromis mossambicus brain cell line (OmB) cells using a lipid-based transfection system designed for delivery of Cas9/gRNA RNPs

Summary

Introduction

Use of fish in physiology studies is widespread with purposes ranging from production enhancement of economically important species to models of basic vertebrate biology. Compared to other gene targeting methods such as TALENS or Zinc fingers that require complex assembly of many DNA binding domain coding sequences into a v ector[10,11], the Cas[9] nuclease can be directed to a specific locus of the genome by merely changing the 5′ terminal ~ 20 bp of a 90 bp RNA molecule (guide RNA or gRNA) to be complementary to the target region adjacent to a genomic NGG Protospacer Adjacent Motif (PAM) sequence[12] Utilization of this powerful tool has great potential to benefit cost-efficient and high-throughput mechanistic studies in fish cell lines. Tilapia (percomorpha), are phylogenetically distant from zebrafish (otomorpha) and tetrapod vertebrates (tetrapodamorpha)[25,26,27], which may render the aforementioned promoters ineffective in cells derived from tilapia and other distantly related species

Methods

Results

Conclusion