Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System

Bai, Yichun; He, Li; Li, Pengcheng; Xu, Kun; Shao, Simin; Ren, Chonghua; Liu, Zhongtian; Wei, Zehui; Zhang, Zhiying

doi:10.1534/g3.116.027706

Cited by 25 publications

(25 citation statements)

References 43 publications

(66 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Thus, all used methodological approaches confirmed the correct-ness and effectiveness of the transgene integration. [Bai et al, 2016]. The low effectivity in our case can be explained by several reasons: we were restricted by 50-100 bp sequence of genomic DNA for selection of gRNAs; the area of targeting is the beginning of 3'UTR, which usually has a lower GC content and has a lot of repeated elements; also the transfection efficiency of the DF1 cells with multiple plasmids was less than 50%.…”

Section: Enrichment Of Successfully Edited Cells With Geneticin Selecmentioning

confidence: 85%

“…We used human codon-optimized Cas9 (hCas9) as it has been previously demonstrated that the optimized Cas9 works in chicken cells and there was no need to synthesize chicken codonoptimized nuclease [Bai et al, 2016 Unique 20bp sequences for the selected gRNAs were cloned under human U6 promoter in the plasmid phU6-gRNA (#53188, Addgene). A plasmid pQE30TaqRFP (Evrogen; Moscow, Russia) coding RFP was used for cotransfection as a reporter of the efficiency of DNA delivery to the cells.…”

Section: Construction Of Expression Vectorsmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2018

View full text Add to dashboard Cite

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.

show abstract

Section: Enrichment Of Successfully Edited Cells With Geneticin Selecmentioning

confidence: 85%

Section: Construction Of Expression Vectorsmentioning

confidence: 99%

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

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Early attempts at implementation in avian embryos employed a tetracycline-inducible Cas9 system to perform gene editing of Pax7 using Tol-2 mediated integration at stages after neural tube closure (Véron et al, 2015). While useful, this approach functioned at low efficiency, particularly at early developmental stages (Bai et al, 2016; Oishi et al, 2016; Véron et al, 2015). Since then, several studies performed in other model systems have reported optimizations of individual CRISPR/Cas9 components, improving our understanding of this gene-editing technology (Chen et al, 2013; Doench et al, 2016; Gandhi et al, 2017; Port et al, 2014; Ren et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo

Gandhi

Piacentino

Vieceli

et al. 2017

Developmental Biology

View full text Add to dashboard Cite

The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific.

show abstract

“…This surrogate system was based on using the puromycin resistance gene and EGFP as double reporter genes to enrich cells with mutations induced by CRISPR/Cas9 nucleases in the human and chicken genomes. In contrast, the mutation frequency of chicken PPAR-γ was 60.7% under puromycin selection; however, only a 0.75% mutation efficiency was detected without puromycin selection [15]. Based on this finding, we directly used this reporter system to enrich cells with targeted mutations.…”

Section: Discussionmentioning

confidence: 99%

“…Efficient gene disruptions of PPAR-γ , ATP5E and OVA were obtained in chicken somatic cells DF-1 with an enrichment system [15]. Véron et al .…”

Section: Introductionmentioning

confidence: 99%

Enhancing Targeted Genomic DNA Editing in Chicken Cells Using the CRISPR/Cas9 System

Yang

Guo²,

et al. 2017

PLoS ONE

View full text Add to dashboard Cite

The CRISPR/Cas9 system has enabled highly efficient genome targeted editing for various organisms. However, few studies have focused on CRISPR/Cas9 nuclease-mediated chicken genome editing compared with mammalian genomes. The current study combined CRISPR with yeast Rad52 (yRad52) to enhance targeted genomic DNA editing in chicken DF-1 cells. The efficiency of CRISPR/Cas9 nuclease-induced targeted mutations in the chicken genome was increased to 41.9% via the enrichment of the dual-reporter surrogate system. In addition, the combined effect of CRISPR nuclease and yRad52 dramatically increased the efficiency of the targeted substitution in the myostatin gene using 50-mer oligodeoxynucleotides (ssODN) as the donor DNA, resulting in a 36.7% editing efficiency after puromycin selection. Furthermore, based on the effect of yRad52, the frequency of exogenous gene integration in the chicken genome was more than 3-fold higher than that without yRad52. Collectively, these results suggest that ssODN is an ideal donor DNA for targeted substitution and that CRISPR/Cas9 combined with yRad52 significantly enhances chicken genome editing. These findings could be extensively applied in other organisms.

show abstract

Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System

Cited by 25 publications

References 43 publications

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

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

Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo

Enhancing Targeted Genomic DNA Editing in Chicken Cells Using the CRISPR/Cas9 System

Contact Info

Product

Resources

About