Abstract
Background: CRISPR/Cas9 system is becoming the dominant genome editing 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. Methods: Three gRNAs were chosen to target chicken 3'-untranslated region 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 ( neoR) under cytomegalovirus promoter. Correct integration of the 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). Results: We have established a CRISPR/Cas9 system to target an 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. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.
Highlights
Modified chickens have great potential in agriculture, industry, biological research and pharmaceuticals [Farzaneh et al, 2017; Lyall et al, 2011; Nishijima & Iijima, 2013; Schock et al, 2016]
Homology regions of 999bp and 3093bp for left and right arms respectively were amplified from the genomic DNA of chicken cell line DF1 by PCR, and cloned using MauBI and PmeI restriction sites for the left arm, and SgrDI and AscI for the right arm respectively (DF-1 genome is yet to be sequenced, common chicken genomic data is available here)
Cas9 in complex with one of the three guide RNA (gRNA) made cuts and produced lengths of cleaved products corresponded to the expected lengths (Figure S3)
Summary
Modified chickens have great potential in agriculture, industry, biological research and pharmaceuticals [Farzaneh et al, 2017; Lyall et al, 2011; Nishijima & Iijima, 2013; Schock et al, 2016]. Precise and effective genome editing is one of the most important aspects in creating genetically modified organisms. Nuclease-mediated gene insertion is several orders of magnitude more efficient compared with spontaneous recombination of DNA template alone [Lin et al, 2014; Zhang et al, 2017] that makes CRISPR/Cas an effective tool for genome editing. CRISPR/Cas mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. Results: We have established a CRISPR/Cas system to target an endogenous locus and precisely insert a gene under endogenous control. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features
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