2019 ASHS Annual Conference

The need for gene edited plants to combat issues of growing population rate, extreme weather, and reduced agricultural land availability is more evident than ever as growers are struggling to adapt to the changing environment. 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, gene editing efficiency of CRISPR/Cas9 in plant relies on the efficacy of generating transgenic plant or tissues with high expression of Cas9 endonuclease. Here, we have constructed a CRISPR-Cas9 vector containing a fused NPT-GFP for high-efficiency gene-editing in annual flowering plants. The high expression of GFP during plant regeneration allowed us to minimize the positional effect on T-DNA expression and preferentially select transgenic seedlings with high expression of Cas9. With this construct, we have targeted the variegation gene LsVar2 in lettuce. Forty independent lines were generated, 27 of which showed strong GFP signals. None of transgenic lines with no GFP signals, although these lines carried T-DNA insertions, exhibited phenotypic difference in leaves compared to the control lines, indicating that GFP signal is at least partially associated with the gene editing events. Six of 27 T0 transgenic lines with GFP signals exhibited variegated leaves. Albino seedlings were observed in segregated progenies of these variegated lines, and sequencing results revealed that homozygous mutations created by Cas9 editing occurred to all these albino seedlings. By contrast, heterozygous mutations were only identified in the plants with variegated leaves and significantly reduced chlorophyll content, and these plants were able to grow and mature normally. Therefore, in addition to confirming the efficiency of the CRISPR-Cas9 vector, our data also provide a proof-of-concept for targeting VAR2 gene in ornamental plants where variegation phenotype is highly favorable. In conclusion, the unique designing of our CRISPR/Cas9 construct allow us to improve gene-editing efficiency and efficiently screen non-TDNA mutants through detecting GFP signals during plant regeneration and progeny segregation.