Mimicking natural polymorphism in eIF4E by CRISPR‐Cas9 base editing is associated with resistance to potyviruses

Abstract

Summary In many crop species, natural variation in eIF 4E proteins confers resistance to potyviruses. Gene editing offers new opportunities to transfer genetic resistance to crops that seem to lack natural eIF 4E alleles. However, because eIF 4E are physiologically important proteins, any introduced modification for virus resistance must not bring adverse phenotype effects. In this study, we assessed the role of amino acid substitutions encoded by a Pisum sativum eIF 4E virus‐resistance allele (W69L, T80D S81D, S84A, G114R and N176K) by introducing them independently into the Arabidopsis thaliana eIF 4E1 gene, a susceptibility factor to the Clover yellow vein virus (Cl YVV ). Results show that most mutations were sufficient to prevent Cl YVV accumulation in plants without affecting plant growth. In addition, two of these engineered resistance alleles can be combined with a loss‐of‐function eIF iso4E to expand the resistance spectrum to other potyviruses. Finally, we use CRISPR ‐ nC as9‐cytidine deaminase technology to convert the Arabidopsis eIF 4E1 susceptibility allele into a resistance allele by introducing the N176K mutation with a single‐point mutation through C‐to‐G base editing to generate resistant plants. This study shows how combining knowledge on pathogen susceptibility factors with precise genome‐editing technologies offers a feasible solution for engineering transgene‐free genetic resistance in plants, even across species barriers.

Prime editing is achievable in the tetraploid potato, but needs improvement

“…Here, we show for the first time that PPE2 strategy developed for dicot species is able to edit an endogenous gene in the cultivated tetraploid potato. Compared to our previous work on cytosine base editing in potato and Arabidopsis (Bastet et al, 2019;Veillet et al, 2019b;Veillet et al, 2019a;Veillet et al, 2020), the efficiency of our PPE strategy appears to be limited, as already observed for cereals. Once improved, the prime editing technology, by largely expanding the range of targeted base substitutions, could make precision breeding amenable in polyploid and vegetatively propagated crops such as potato.…”

Mining downy mildew susceptibility genes: a diversity study in grapevine

“…The detection of specific homozygous variants in the natural pool can guide genome editing projects in targeting the “naturally” occurring mutations. This “tailored gene editing” mimicking natural polymorphisms, has been recently demonstrated by Bastet et al (2017; 2019). Moreover, breeding programs could take advantage of the information on homozygous and heterozygous selected mutations of S-genes in a next-generation marker-assisted breeding program.…”

Deploying Genome Editing Tools for Dissecting the Biology of Nut Trees

“…Here we presented the first report to our knowledge of gene-editing using Cas9 in a nut crop. The versatility of CRISPR-Cas9 tools goes beyond point mutations adjacent to PAM sites, with options in base-editing variations already employed in some crops (Bastet et al, 2019;Wu et al, 2019). When used in combination, multiple sgRNAs targeting the same exon or different exons within the same gene provide a higher chance of a functional knockout (Xie et al, 2015).…”