Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line

Abstract

CRISPR/Cas9 system is becoming the dominant genome editing Background: tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific.Three gRNAs were chosen to target chicken 3'-untranslated region Methods: of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( ) under cytomegalovirus promoter. Correct integration of the neoR cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS).We have established a CRISPR/Cas9 system to target an Results: endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter.The approach can be used further to insert genes of interest Conclusions: under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

Optimized CRISPR/Cas9 system for gene knockout in chicken DF1 cells

“…However, our results showed that the T7E1 assay almost couldn't detect positive cells in the groups without puromycin selection. In the groups with puromycin selection, the T7E1 assay detected 12.9–72.1% indels, which was similar to the results of the previous reports ( Abu-Bonsrah et al, 2016 ; Bai et al, 2016 ; Antonova et al, 2018 ). The performance of T7E1 may be impacted by the length and identity of base pair mismatches, flanking sequence, secondary structure, and relative abundance of mutant sequence ( Vouillot et al, 2015 ; Michalski et al, 2021 ).…”

“…In general, affected by transfection, cleavage, and repair efficiencies of the CRISPR/Cas9 system, most cells will not be induced with intent mutations at target sites ( Kim et al, 2011 ; Ren et al, 2015 ). Thus, simple methods for enriching cells with desired genetic modification are essential to facilitate genome editing in living cells ( Yan et al, 2016 ; Zhou et al, 2016 ; Antonova et al, 2018 ). For this approach, we used an RPG surrogate reporter in the present study based on antibiotic selection to enrich cells with gene mutations.…”