Recovery of the non-functional EGFP-assisted identification of mutants generated by CRISPR/Cas9.

Abstract

The recovery of non-functional-enhanced green fluorescence protein can be used as indicator to facilitate the identification of mutants generated by CRISPR/Cas9. The CRISPR/Cas9 system is a powerful tool for genome editing and it has been employed to knock out genes of interest in multiple plant species. Identification of desired mutants from regenerated plants is necessary prior to functional study. Current screening methods work based on the purification of genomic DNA and it would be laborious and time consuming using these methods to screen mutants from a large population of seedlings. Here, we developed the non-functional enhanced green fluorescence protein (nEGFP) reporter gene by inserting a single guide RNA (sgRNA) and the protospacer adjacent motif in the 5' coding region of EGFP, and the activity of nEGFP could be recovered after successful targeted editing. Using the nEGFP as the reporter gene in Nicotiana tabacum, we found that over 94% of the plants exhibiting EGFP fluorescence were confirmed to be desired mutants. The use of this nEGFP reporter construct had limited negative effect on editing efficiency, and the expression of Cas9 and sgRNA was not affected. Moreover, this method was also applied in grape by targeting the phytoene desaturase gene(PDS), and the grape cells with EGFP signal were revealed to contain targeted mutations in VvPDS. Our results show that the nEGFP gene can be used as reporter to help screen mutants according to the recovered EGFP fluorescence during the application of CRISPR/Cas9 in plants.