GFP tagging based method to analyze the genome editing efficiency of CRISPR/Cas9-gRNAs through transient expression in N. benthamiana
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The CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR—associated proteins 9) is simple and highly efficient technology applied to functional studies of genes and genetic crop improvement. In this study, we have demonstrated the utility of green fluorescent protein (GFP) marker to detect the targeting efficiency of gRNAs. As a proof of concept, Glycine max De-Etiolated 1 (GmDET1) gene was chosen and tagged with GFP to rapidly analyze genome editing efficiency of gRNAs. Results showed weaker GFP fluorescence signal in the N. benthamiana leaves co-infiltrated with GmDET1-GFP overexpression (OE) + DET1 gRNA1 constructs as compared to the stronger GFP florescence signal in the leaves co-infiltrated with DET1 gRNA2 and gRNA3 constructs, thus indicating the highest of DET1 gRNA1. These results were further confirmed by the detection of the mutation frequencies through T7 endonuclease (T7E1) assay and sequencing; the highest mutation rate of 38.46% in GmDET1 targeted by DET1 gRNA1 to that of DET1 gRNA2 (7.69%) and gRNA3 (15.38%) was observed. Thus our studies showed “GFP tagging” as the most reliable and rapid method-one can apply to minimize the generation of non-edited transgenic plants resulting from inefficient gRNAs.
KeywordsCRISPR/Cas9 gRNA GFP fluorescent marker N. benthamiana Glycine max DET1
Clustered regularly interspaced short palindromic repeats/CRISPR—associated proteins 9
Green fluorescent protein
T7 Endonuclease I
We thank the laboratory of Robert Stupar for Cas9 (MDC123) and gRNA shuttle vectors (pBlu/gRNA) (Addgene plasmid # 59184 and 59188 respectively). Permission was taken to conduct this study from Institute Biosafety Committee. The presented research work was carried out with the financial support received from ICAR-IARI, Division of Biochemistry, New Delhi-110012.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
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