We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

BackgroundThe use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA.ResultsHere, we use geminivirus replicons to create heritable modifications to the tomato genome at frequencies tenfold higher than traditional methods of DNA delivery (i.e., Agrobacterium). A strong promoter was inserted upstream of a gene controlling anthocyanin biosynthesis, resulting in overexpression and ectopic accumulation of pigments in tomato tissues. More than two-thirds of the insertions were precise, and had no unanticipated sequence modifications. Both TALENs and CRISPR/Cas9 achieved gene targeting at similar efficiencies. Further, the targeted modification was transmitted to progeny in a Mendelian fashion. Even though donor molecules were replicated in the vectors, no evidence was found of persistent extra-chromosomal replicons or off-target integration of T-DNA or replicon sequences.ConclusionsHigh-frequency, precise modification of the tomato genome was achieved using geminivirus replicons, suggesting that these vectors can overcome the efficiency barrier that has made gene targeting in plants challenging. This work provides a foundation for efficient genome editing of crop genomes without the random integration of foreign DNA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0796-9) contains supplementary material, which is available to authorized users.

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Sex chromosomes have repeatedly evolved from a pair of autosomes. Consequently, X and Y chromosomes initially have similar gene content, but ongoing Y degeneration leads to reduced expression and eventual loss of Y genes. The resulting imbalance in gene expression between Y genes and the rest of the genome is expected to reduce male fitness, especially when protein networks have components from both autosomes and sex chromosomes. A diverse set of dosage compensating mechanisms that alleviates these negative effects has been described in animals. However, the early steps in the evolution of dosage compensation remain unknown, and dosage compensation is poorly understood in plants. Here, we describe a dosage compensation mechanism in the evolutionarily young XY sex determination system of the plant Silene latifolia. Genomic imprinting results in higher expression from the maternal X chromosome in both males and females. This compensates for reduced Y expression in males, but results in X overexpression in females and may be detrimental. It could represent a transient early stage in the evolution of dosage compensation. Our finding has striking resemblance to the first stage proposed by Ohno for the evolution of X inactivation in mammals.

Structurally and functionally diverged sex chromosomes have evolved in many animals as well as in some plants. Sex chromosomes represent a specific genomic region(s) with locally suppressed recombination. As a consequence, repetitive sequences involving transposable elements, tandem repeats (satellites and microsatellites), and organellar DNA accumulate on the Y (W) chromosomes. In this paper, we review the main types of repetitive elements, their gathering on the Y chromosome, and discuss new findings showing that not only accumulation of various repeats in non-recombining regions but also opposite processes form Y chromosome. The aim of this review is also to discuss the mechanisms of repetitive DNA spread involving (retro) transposition, DNA polymerase slippage or unequal crossing-over, as well as modes of repeat removal by ectopic recombination. The intensity of these processes differs in non-recombining region(s) of sex chromosomes when compared to the recombining parts of genome. We also speculate about the relationship between heterochromatinization and the formation of heteromorphic sex chromosomes.