Cut DNA ends in plants may recombine to form novel molecules. We asked whether CRISPR-Cas9 expression in plants could induce nonhomologous recombination between diverse and heterologous broken DNA ends. We induced two breaks separated by 2.3 or by 8.5 kilobases leading to duplication of the intervening DNA and meiotic transmission of the 2.3kb duplication. Two or more dsDNA breaks in nonhomologous chromosomes led to ligation of breakpoints consistent with chromosome arm translocations. Screening 881 primary transformants we obtained 195 PCR products spanning independent, expected translocation junctions involving ends produced by cutting different loci. Sequencing indicated a true positive rate of 84/91 and demonstrated the occurrence of different junction alleles. A majority of the resulting structures would be deleterious and none were transmitted meiotically. Ligation of interchromosomal, heterologous dsDNA ends suggest that the CRISPR-Cas9 can be used to engineer plant genes and chromosomes in vivo.Significance StatementWe explored how genome editing tools such as CRISPR-Cas9 could provide new ways to tailor novel genomic combinations and arrangements. We show that distant cut ends often precisely come together, that cuts in different chromosomes can result in translocations, and that two cuts within a chromosome often result in the duplication of the intervening segment. Formation of multiple structures with precise junctions will enable engineered rearrangements that can be predicted with accuracy.
Uniparental genome elimination is a dramatic case of centromeric failure, resulting in the postzygotic loss of a parental chromosome set. Genome partitioning during cell division relies on centromere attachment to spindle fibers through kinetochores. Centromeres are epigenetically specified by CENP-A (CENH3), a conserved centromeric specific histone H3 variant. In Arabidopsis, CENH3 modification results in haploid inducers, whose genome is eliminated frequently when crossed to the wild type. To investigate the underlying mechanism, we dissected the timing and molecular features of genome elimination. In zygotes and early embryos from genome elimination crosses, CENH3 occupied only the centromeres contributed by the wild-type parent. Haploid inducer chromosomes had defective kinetochores and missegregated, often forming micronuclei. This uniparental loss of centromere identity is initiated by the removal of altered CENH3 at fertilization, while wild-type CENH3 persists and maintains strong centromeric identity. Weak centromeres were capable of rebuilding functional kinetochores, but often failed when in competition with normal ones. We induced a similar weak state by mitotic dilution of wild-type CENH3. Furthermore, weakness was suppressed by crosses of haploid inducers to other variants of haploid inducers, and enhanced by mutations in VIM1, a ubiquitin ligase known to modify CENH3 and centromeric DNA methylation.. The differential stability of altered CENH3 during reproduction has important genetic and evolutionary implications.
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