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

Authors: NGUYEN, CHI - University Of Florida; LI, JUNCHENG - University Of Florida; Mou, Beiquan; GONG, HAIJUN - Northwest Agriculture And Forestry University; HUO, HEQIANG - University Of Florida

Submitted to: Vegetable Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/10/2021
Publication Date: 5/25/2021
Citation: Nguyen, C.D., Li, J., Mou, B., Gong, H., Huo, H. 2021. A case study of using an efficient CRISPR/Cas9 system to develop variegated lettuce. Vegetable Research. 1. Article 4. https://doi.org/10.48130/VR-2021-0004.
DOI: https://doi.org/10.48130/VR-2021-0004

Interpretive Summary: The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR 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 encoding the green fluorescent protein so we can visually select transgenic plants, thus saving time and resources. Successful gene editing using CRISPR/Cas9 has been illustrated in different plant species. However, an important aesthetic characteristic such as leaf variegation remained unexplored. With the newly designed method, we have targeted the variegation gene LsVar2 in lettuce for editing and created variegated lettuce leaves. In conclusion, the unique design of the new method allow us to improve gene-editing efficiency and effectively screen mutants visually during plant regeneration and in progeny plants. Additionally, we used lettuce variegation as a proof of concept to develop new variegated plant breeding materials for ornamental plant species.

Technical Abstract: The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR 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 NPT-GFP where the high expression of GFP during plant regeneration allowed us to minimize the positional effect on T-DNA expression and preferentially select transgenic plants with high expression of Cas9. Additionally, segregation of T-DNA and mutations can be visually tracked with GFP signal thus saving time and resources. Successful gene editing using CRISPR/Cas9 has been illustrated in different plant species. However, an important aesthetic characteristic such as leaf variegation remained unexplored. With the newly designed construct, we have targeted the variegation gene LsVar2 in lettuce. Our results indicated that LsVAR2 is closely related to both AtFtsH2 and AtFtsH8 where homozygous mutant lead to an albino phenotype while variegated phenotype was induced by CRISPR/Cas9 de novo gene editing. In conclusion, the unique design of our CRISPR/Cas9 construct allowed us to improve gene-editing efficiency and effectively screen non-TDNA mutants through detection of GFP signals during plant regeneration and progeny segregation. Additionally, we provided lettuce variegation as a proof of method to develop new variegated plant breeding materials for ornamental plant species.