Efficient genome editing using CRISPR/Cas9 ribonucleoprotein approach in cultured medaka fish cells

Abstract

Gene editing with CRISPR/Cas9 is a powerful tool to study the function of target genes. Although this technology has demonstrated wide efficiency in many species, including fertilized zebrafish and medaka fish embryos when microinjected, its application to achieve efficient gene editing in cultured fish cells have met some difficulty. Here, we report an efficient and reliable approach to edit genes in cultured medaka (Oryzias latipes) fish cells using pre-formed gRNA-Cas9 ribonucleoprotein (RNP) complex. Both medaka fish haploid and diploid cells were transfected with the RNP complex by electroporation. Efficient gene editing was demonstrated by polymerase chain reaction (PCR) amplification of the target gene from genomic DNA and heteroduplex mobility assay carried out with polyacrylamide gel electrophoresis (PAGE). The heteroduplex bands caused by RNP cleavage and non-homologous end joining could be readily detected by PAGE. DNA sequencing confirmed that these heteroduplex bands contains the mutated target gene sequence. The average gene editing efficiency in haploid cells reached 50%, enabling us to generate a clonal cell line with ntrk3b gene mutation for further study. This RNP transfection method also works efficiently in diploid medaka cells, with the highest mutation efficiency of 61.5%. The specificity of this synthetic RNP CRISPR/Cas9 approach was verified by candidate off-target gene sequencing. Our result indicated that transfection of pre-formed gRNA-Cas9 RNP into fish cells is efficient and reliable to edit target genes in cultured medaka fish cells. This method will be very useful for gene function studies using cultured fish cells.

Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes

“…'Edit' refers to the estimated percentage of edited cells, while 'KO' refers to the estimated percentage of cells which contain edits expected to result in GFP knockout electroporation of Cas9 or Cas12a RNP. The method can be used to edit over 90% of cells in a mixed cell line population with Cas9, and over 60% with Cas12a; this is generally a higher editing efficiency than reported in medaka fish cell lines using Cas9 RNP (62% editing, Liu et al 2018). The optimised RNP method circumvents several challenges to genetic engineering of many fish cell lines due to their slow growth, poor transfection/transduction efficiency, and difficulty to obtain clonal lines (Collet et al 2018).…”

“…Indeed lentivirus transduction using the approach of Gratacap et al ( 2020 ) resulted in a very low success rate in the Atlantic salmon SHK-1 cell line (< 1% of cells successfully transduced; data not shown). CRISPR/Cas ribonucleoprotein (RNP) complexes have potential for editing of fish cell lines, as demonstrated with efficiency of up to 62% in medaka ( Oryzias latipes ) (Liu et al 2018 ). Furthermore, Cas12a editing has been successfully applied in mammalian cells, Xenopus and zebrafish ( Danio rerio ), including using RNP systems (Moreno-Mateos et al 2017 ; Liu et al 2019 ), and significantly expands the number of ‘editable’ sites in the target species’ genomes.…”

PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts

“…Its viability and concentration were determined using Fluorescein Diacetate (FDA; 0.2%) dye in the hemocytometer. To confirm the internalization of the RNP complex inside cells, an assay was performed using fluorescent-labeled tracrRNA molecules (ATTO 550, IDT) [ 9 ]. Microphotographs were taken using an inverted optic microscope ix80 Olympus.…”

“…CRISPR-Cas9 technology can be used as RNP complexes without the introgression and expression of a transgenic cassette in the host genome [ 8 ]. Such an approach would avoid a number of generations of backcrossing, expression vectors, and other invasive methods of cell penetration (e.g., biolistics) that can lead to gene disruption, including large deletions, partial trisomy, genome shattering events, and plant mosaicism [ 9 ]. Overall, these side effects can mask or interfere in the target gene functional analysis, and further additional biosafety concerns prior to commercial release.…”

Efficient Crispr-cas9 Mediated Pooled-sgRNAs&nbsp;Assembly Accelerates Targeting Multiple Genes Related to Male Sterility in Cotton&nbsp;

“…This system contains a powerful, precious and effective nuclease (Cas9) that is activated by a guided-RNA to induce different type of targeted mutations: deletion, insertion, substitutions; resulting in disabling gene function. Owing to its simplicity and high e ciency, CRISPR-Cas9 technology is harnessed to make precise changes in almost whole living specie's genome like human cells, fruit ies, sh, mice, and plants [21][22][23][24][25][26].…”