CRISPR/Cas9-induced DNA breaks trigger crossover, chromosomal loss, and chromothripsis-like rearrangements.

DNA double-stranded breaks (DSBs) generated by the Cas9 nuclease are commonly repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR). However, little is known about unrepaired DSBs and the type of damage they trigger in plants. We designed an assay that detects loss of heterozygosity (LOH) in somatic cells, enabling the study of a broad range of DSB-induced genomic events. The system relies on a mapped phenotypic marker which produces a light purple color (betalain pigment) in all plant tissues. Plants with sectors lacking the Betalain marker upon DSB induction between the marker and the centromere were tested for LOH events. Using this assay, we detected a tomato (Solanum lycopersicum) flower with a twin yellow and dark purple sector, corresponding to a germinally transmitted somatic crossover event. We also identified instances of small deletions of genomic regions spanning the T-DNA and whole chromosome loss. In addition, we show that major chromosomal rearrangements including loss of large fragments, inversions, and translocations were clearly associated with the CRISPR-induced DSB. Detailed characterization of complex rearrangements by whole-genome sequencing and molecular and cytological analyses supports a model in which a breakage-fusion-bridge cycle followed by chromothripsis-like rearrangements had been induced. Our LOH assay provides a tool for precise breeding via targeted crossover detection. It also uncovers CRISPR-mediated chromothripsis-like events in plants.

Mutagenesis of the melon Prv gene by CRISPR/Cas9 breaks papaya ringspot virus resistance and generates an autoimmune allele with constitutive defense responses.

The majority of plant disease resistance (R) genes encode nucleotide binding-leucine-rich repeat (NLR) proteins. In melon, two closely linked NLR genes, Fom-1 and Prv, were mapped and identified as candidate genes that control resistance to Fusarium oxysporum f.sp. melonis races 0 and 2, and to papaya ringspot virus (PRSV), respectively. In this study, we validated the function of Prv and showed that it is essential for providing resistance against PRSV infection. We generated CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] mutants using Agrobacterium-mediated transformation of a PRSV-resistant melon genotype, and the T1 progeny proved susceptible to PRSV, showing strong disease symptoms and viral spread upon infection. Three alleles having 144, 154, and ~3 kb deletions, respectively, were obtained, all of which caused loss of resistance. Interestingly, one of the Prv mutant alleles, prvΔ154, encoding a truncated product, caused an extreme dwarf phenotype, accompanied by leaf lesions, high salicylic acid levels, and defense gene expression. The autoimmune phenotype observed at 25 °C proved to be temperature dependent, being suppressed at 32 °C. This is a first report on the successful application of CRISPR/Cas9 to confirm R gene function in melon. Such validation opens up new opportunities for molecular breeding of disease resistance in this important vegetable crop.

CRISPR/Cas9 Induced Somatic Recombination at the CRTISO Locus in Tomato.

Homologous recombination (HR) in somatic cells is not as well understood as meiotic recombination and is thought to be rare. In a previous study, we showed that Inter-Homologous Somatic Recombination (IHSR) can be achieved by targeted induction of DNA double-strand breaks (DSBs). Here, we designed a novel IHSR assay to investigate this phenomenon in greater depth. We utilized F1 hybrids from divergent parental lines, each with a different mutation at the Carotenoid isomerase (CRTISO) locus. IHSR events, namely crossover or gene conversion (GC), between the two CRTISO mutant alleles (tangerine color) can restore gene activity and be visualized as gain-of-function, wildtype (red) phenotypes. Our results show that out of four intron DSB targets tested, three showed DSB formation, as seen from non-homologous end-joining (NHEJ) footprints, but only one target generated putative IHSR events as seen by red sectors on tangerine fruits. F2 seeds were grown to test for germinal transmission of HR events. Two out of five F1 plants showing red sectors had their IHSR events germinally transmitted to F2, mainly as gene conversion. Six independent recombinant alleles were characterized: three had truncated conversion tracts with an average length of ~1 kb. Two alleles were formed by a crossover as determined by genotyping and characterized by whole genome sequencing. We discuss how IHSR can be used for future research and for the development of novel gene editing and precise breeding tools.

Redistribution of Meiotic Crossovers Along Wheat Chromosomes by Virus-Induced Gene Silencing.

Meiotic recombination is the main driver of genetic diversity in wheat breeding. The rate and location of crossover (CO) events are regulated by genetic and epigenetic factors. In wheat, most COs occur in subtelomeric regions but are rare in centromeric and pericentric areas. The aim of this work was to increase COs in both "hot" and "cold" chromosomal locations. We used Virus-Induced gene Silencing (VIGS) to downregulate the expression of recombination-suppressing genes XRCC2 and FANCM and of epigenetic maintenance genes MET1 and DDM1 during meiosis. VIGS suppresses genes in a dominant, transient and non-transgenic manner, which is convenient in wheat, a hard-to-transform polyploid. F1 hybrids of a cross between two tetraploid lines whose genome was fully sequenced (wild emmer and durum wheat), were infected with a VIGS vector ∼ 2 weeks before meiosis. Recombination was measured in F2 seedlings derived from F1-infected plants and non-infected controls. We found significant up and down-regulation of CO rates along subtelomeric regions as a result of silencing either MET1, DDM1 or XRCC2 during meiosis. In addition, we found up to 93% increase in COs in XRCC2-VIGS treatment in the pericentric regions of some chromosomes. Silencing FANCM showed no effect on CO. Overall, we show that CO distribution was affected by VIGS treatments rather than the total number of COs which did not change. We conclude that transient silencing of specific genes during meiosis can be used as a simple, fast and non-transgenic strategy to improve breeding abilities in specific chromosomal regions.

Single Molecule Imaging of T-DNA Intermediates Following Agrobacterium tumefaciens Infection in Nicotiana benthamiana.

Plant transformation mediated by Agrobacterium tumefaciens is a well-studied phenomenon in which a bacterial DNA fragment (T-DNA), is transferred to the host plant cell, as a single strand, via type IV secretion system and has the potential to reach the nucleus and to be integrated into its genome. While Agrobacterium-mediated transformation has been widely used for laboratory-research and in breeding, the time-course of its journey from the bacterium to the nucleus, the conversion from single- to double-strand intermediates and several aspects of the integration in the genome remain obscure. In this study, we sought to follow T-DNA infection directly using single-molecule live imaging. To this end, we applied the LacO-LacI imaging system in Nicotiana benthamiana, which enabled us to identify double-stranded T-DNA (dsT-DNA) molecules as fluorescent foci. Using confocal microscopy, we detected progressive accumulation of dsT-DNA foci in the nucleus, starting 23 h after transfection and reaching an average of 5.4 and 8 foci per nucleus at 48 and 72 h post-infection, respectively. A time-course diffusion analysis of the T-DNA foci has demonstrated their spatial confinement.

Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system.

Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon, we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene, via homologous recombination (HR). Mutagenesis experiments, performed in the Micro-Tom variety, achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting (GT) experiments, our target was a fast-neutron-induced crtiso allele that contained a 281-bp deletion. This deletion was repaired with the wild-type sequence through HR between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient GT can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops.