A case study of using an efficient CRISPR/Cas9 system to develop variegated lettuce

Abstract

The clustered, regularly interspaced, short palindromic repeat associated endonuclease 9 (CRISPR/Cas9) system has emerged as a powerful approach for precision breeding to create plants with desirable traits. However, the CRISPR/Cas9 system relies heavily on an efficient plant transformation system that is usually time-consuming and costly. Here, we have constructed a CRISPR-Cas9 vector with neomycin phosphotransferase II and green fluorescent protein (eGFP-NPTII), where the high expression of GFP during plant regeneration allowed us to minimize the positional effect on T-DNA expression and facilitate screening T-DNA-free mutants. Successful gene editing using CRISPR/Cas9 has been illustrated in different plant species, but an important aesthetic characteristic of leaf variegation remained unexplored. With the newly designed construct, we have targeted the variegation gene <i>LsVAR2</i> in lettuce. Our results indicated that <i>LsVAR2</i> is closely related to both <i>AtFtsH2</i> and <i>AtFtsH8</i>, in which homozygous mutations lead to an albino phenotype while a variegated phenotype was induced by CRISPR/Cas9 <i>de novo</i> gene editing. In conclusion, the unique design of our CRISPR/Cas9 construct could efficiently edit the target gene and ease the screening of non-TDNA mutants through detecting GFP signals during plant regeneration and progeny segregation. Additionally, the success of gene-editing of <i>LsVAR2</i> in lettuce demonstrates proof in this method to develop novel plant breeding materials for valuable horticultural plant species.