An iterative gene-editing strategy broadens eIF4E1 genetic diversity in Solanum lycopersicum and generates resistance to multiple potyvirus isolates

International audienceResistance to potyviruses in plants has been largely provided by the selection of natural variant alleles of eukaryotic translation initiation factors (eIF) 4E in many crops. However, the sources of such variability for breeding can be limited for certain crop species, while new virus isolates continue to emerge. Different methods of mutagenesis have been applied to inactivate the eIF4E genes to generate virus resistance, but with limited success due to the physiological importance of translation factors and their redundancy. Here, we employed genome editing approaches at the base level to induce non-synonymous mutations in the eIF4E1 gene and create genetic diversity in cherry tomato (Solanum lycopersicum var. cerasiforme). We sequentially edited the genomic sequences coding for two regions of eIF4E1 protein, located around the cap-binding pocket and known to be important for susceptibility to potyviruses. We show that the editing of only one of the two regions, by gene knock-in and base editing, respectively, is not sufficient to provide resistance. However, combining amino acid mutations in both regions resulted in resistance to multiple potyviruses without affecting the functionality in translation initiation. Meanwhile, we report that extensive base editing in exonic region can alter RNA splicing pattern, resulting in gene knockout. Altogether our work demonstrates that precision editing allows to design plant factors based on the knowledge on evolutionarily selected alleles and enlarge the gene pool to potentially provide advantageous phenotypes such as pathogen resistance

TRAIL receptor gene editing unveils TRAIL-R1 as a master player of apoptosis induced by TRAIL and ER stress

TRAIL induces selective tumor cell death through TRAIL-R1 and TRAIL-R2. Despite the fact that these receptors share high structural homologies, induction of apoptosis upon ER stress, cell autonomous motility and invasion have solely been described to occur through TRAIL-R2. Using the TALEN gene-editing approach, we show that TRAIL-R1 can also induce apoptosis during unresolved unfolded protein response (UPR). Likewise, TRAIL-R1 was found to co-immunoprecipitate with FADD and caspase-8 during ER stress. Its deficiency conferred resistance to apoptosis induced by thaspigargin, tunicamycin or brefeldin A. Our data also demonstrate that tumor cell motility and invasion-induced by TRAIL-R2 is not cell autonomous but induced in a TRAIL-dependant manner. TRAIL-R1, on the other hand, is unable to trigger cell migration owing to its inability to induce an increase in calcium flux. Importantly, all the isogenic cell lines generated in this study revealed that apoptosis induced TRAIL is preferentially induced by TRAIL-R1. Taken together, our results provide novel insights into the physiological functions of TRAIL-R1 and TRAIL-R2 and suggest that targeting TRAIL-R1 for anticancer therapy is likely to be more appropriate owing to its lack of pro-motile signaling capability

Trail receptor gene editing unveils trail-r1 as a master player of apoptosis induced by trail and er stres

TRAIL induces selective tumor cell death through TRAIL-R1 and TRAIL-R2. Despite the fact that these receptors share high structural homologies, induction of apoptosis upon ER stress, cell autonomous motility and invasion have solely been described to occur through TRAIL-R2. Using the TALEN gene-editing approach, we show that TRAIL-R1 can also induce apoptosis during unresolved unfolded protein response (UPR). Likewise, TRAIL-R1 was found to co-immunoprecipitate with FADD and caspase-8 during ER stress. Its deficiency conferred resistance to apoptosis induced by thaspigargin, tunicamycin or brefeldin A. Our data also demonstrate that tumor cell motility and invasion-induced by TRAIL-R2 is not cell autonomous but induced in a TRAIL-dependant manner. TRAIL-R1, on the other hand, is unable to trigger cell migration owing to its inability to induce an increase in calcium flux. Importantly, all the isogenic cell lines generated in this study revealed that apoptosis induced TRAIL is preferentially induced by TRAIL-R1. Taken together, our results provide novel insights into the physiological functions of TRAIL-R1 and TRAIL-R2 and suggest that targeting TRAIL-R1 for anticancer therapy is likely to be more appropriate owing to its lack of pro-motile signaling capability

Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤0.1% to ∼6%) with that of homologous gene targeting (≤0.1% to ∼15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering