Expanding the CRISPR Toolbox in P. patens Using SpCas9-NG Variant and Application for Gene and Base Editing in Solanaceae Crops.

Genome editing has become a major tool for both functional studies and plant breeding in several species. Besides generating knockouts through the classical CRISPR-Cas9 system, recent development of CRISPR base editing holds great and exciting opportunities for the production of gain-of-function mutants. The PAM requirement is a strong limitation for CRISPR technologies such as base editing, because the base substitution mainly occurs in a small edition window. As precise single amino-acid substitution can be responsible for functions associated to some domains or agronomic traits, development of Cas9 variants with relaxed PAM recognition is of upmost importance for gene function analysis and plant breeding. Recently, the SpCas9-NG variant that recognizes the NGN PAM has been successfully tested in plants, mainly in monocotyledon species. In this work, we studied the efficiency of SpCas9-NG in the model moss Physcomitrella patens and two Solanaceae crops (Solanum lycopersicum and Solanum tuberosum) for both classical CRISPR-generated gene knock-out and cytosine base editing. We showed that the SpCas9-NG greatly expands the scope of genome editing by allowing the targeting of non-canonical NGT and NGA PAMs. The CRISPR toolbox developed in our study opens up new gene function analysis and plant breeding perspectives for model and crop plants.

Transgene-Free Genome Editing in Tomato and Potato Plants Using Agrobacterium-Mediated Delivery of a CRISPR/Cas9 Cytidine Base Editor.

Genome editing tools have rapidly been adopted by plant scientists for gene function discovery and crop improvement. The current technical challenge is to efficiently induce precise and predictable targeted point mutations valuable for crop breeding purposes. Cytidine base editors (CBEs) are CRISPR/Cas9 derived tools recently developed to direct a C-to-T base conversion. Stable genomic integration of CRISPR/Cas9 components through Agrobacterium-mediated transformation is the most widely used approach in dicotyledonous plants. However, elimination of foreign DNA may be difficult to achieve, especially in vegetatively propagated plants. In this study, we targeted the acetolactate synthase (ALS) gene in tomato and potato by a CBE using Agrobacterium-mediated transformation. We successfully and efficiently edited the targeted cytidine bases, leading to chlorsulfuron-resistant plants with precise base edition efficiency up to 71% in tomato. More importantly, we produced 12.9% and 10% edited but transgene-free plants in the first generation in tomato and potato, respectively. Such an approach is expected to decrease deleterious effects due to the random integration of transgene(s) into the host genome. Our successful approach opens up new perspectives for genome engineering by the co-edition of the ALS with other gene(s), leading to transgene-free plants harboring new traits of interest.

Systematic silencing of a tobacco nitrate reductase transgene in lettuce (Lactuca sativa L.).

A population of 50 independent transgenic lettuces transformed with a nitrate reductase coding sequence under the control of the 35S promoter was studied. None of them showed significantly lower nitrate levels when compared with the untransformed plants, despite the presence of nitrate reductase (NR) activity that derives from the transgene in at least four of the transformants. No repercussion on total NR activity (endogenous+transgenic) was detected in these plants. Nevertheless, 28% of the transformants showed phenotypes characteristic of a general silencing of the NR genes as already described in tobacco and potato, i.e. bleaching of the leaves leading to the death of the plant. By northern blots, it was shown that the transgene was silenced in these chlorotic plants and also in the plants that did not show symptoms of chlorosis. Thus a silencing process specifically directed against the NR mRNA derived from the transgene occurred very early in the development of all the plants studied, whatever homologous endogenous NR mRNA is present in the plant. In some cases this transgene-specific silencing was shown subsequently to extend to the homologous endogenous NR mRNA. These results suggest that, in lettuce, the level of nitrate reductase mRNA is under tight expression control and this is able specifically to target transgenic transcripts by a post-transcriptional gene silencing (PTGS) mechanism during the first stage of development of the plantlet.