Multiplex Cas9-based excision of CLCuV betasatellite and DNA-A revealed reduction of viral load with asymptomatic cotton plants.

MAIN CONCLUSION: Multiplexed Cas9-based genome editing of cotton resulted in reduction of viral load with asymptomatic cotton plants. In depth imaging of proteomic dynamics of resulting CLCuV betasatellite and DNA-A protein was also performed. The notorious  cotton leaf curl virus (CLCuV), which is transmitted by the sap-sucking insect whitefly, continuously damages cotton crops. Although the application of various toxins and RNAi has shown some promise, sustained control has not been achieved. Consequently, CRISPR_Cas9 was applied by designing multiplex targets against DNA-A (AC2 and AC3) and betasatellite (ßC1) of CLCuV using CRISPR direct and ligating into the destination vector of the plant using gateway ligation method. The successful ligation of targets into the destination vector was confirmed by the amplification of 1049 bp using a primer created from the promoter and target, while restriction digestion using the AflII and Asc1 enzymes determined how compact the plasmid developed and the nucleotide specificity of the plasmid was achieved through Sanger sequencing. PCR confirmed the successful introduction of plasmid into CKC-1 cotton variety. Through Sanger sequencing and correlation with the mRNA expression of DNA-A and betasatellite in genome-edited cotton plants subjected to agroinfiltration of CLCuV infectious clone, the effectiveness of knockout was established. The genome-edited cotton plants demonstrated edited efficacy of 72% for AC2 and AC3 and 90% for the (ßC1) through amplicon sequencing, Molecular dynamics (MD) simulations were used to further validate the results. Higher RMSD values for the edited ßC1 and AC3 proteins indicated functional loss caused by denaturation. Thus, CRISPR_Cas9 constructs can be rationally designed using high-throughput MD simulation technique. The confidence in using this technology to control plant virus and its vector was determined by the knockout efficiency and the virus inoculation assay.

Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system.

During the evolution of plant genomes, sequence inversions occurred repeatedly making the respective regions inaccessible for meiotic recombination and thus for breeding. Therefore, it is important to develop technologies that allow the induction of inversions within chromosomes in a directed and efficient manner. Using the Cas9 nuclease from Staphylococcus aureus (SaCas9), we were able to obtain scarless heritable inversions with high efficiency in the model plant Arabidopsis thaliana. Via deep sequencing, we defined the patterns of junction formation in wild-type and in the non-homologous end-joining (NHEJ) mutant ku70-1. Surprisingly, in plants deficient of KU70, inversion induction is enhanced, indicating that KU70 is required for tethering the local broken ends together during repair. However, in contrast to wild-type, most junctions are formed by microhomology-mediated NHEJ and thus are imperfect with mainly deletions, making this approach unsuitable for practical applications. Using egg-cell-specific expression of Cas9, we were able to induce heritable inversions at different genomic loci and at intervals between 3 and 18 kb, in the percentage range, in the T1 generation. By screening individual lines, inversion frequencies of up to the 10% range were found in T2. Most of these inversions had scarless junctions and were without any sequence change within the inverted region, making the technology attractive for use in crop plants. Applying our approach, it should be possible to reverse natural inversions and induce artificial ones to break or fix linkages between traits at will.

From classical mutagenesis to nuclease-based breeding - directing natural DNA repair for a natural end-product.

Production of mutants of crop plants by the use of chemical or physical genotoxins has a long tradition. These factors induce the natural DNA repair machinery to repair damage in an error-prone way. In the case of radiation, multiple double-strand breaks (DSBs) are induced randomly in the genome, leading in very rare cases to a desirable phenotype. In recent years the use of synthetic, site-directed nucleases (SDNs) - also referred to as sequence-specific nucleases - like the CRISPR/Cas system has enabled scientists to use exactly the same naturally occurring DNA repair mechanisms for the controlled induction of genomic changes at pre-defined sites in plant genomes. As these changes are not necessarily associated with the permanent integration of foreign DNA, the obtained organisms per se cannot be regarded as genetically modified as there is no way to distinguish them from natural variants. This applies to changes induced by DSBs as well as single-strand breaks, and involves repair by non-homologous end-joining and homologous recombination. The recent development of SDN-based 'DNA-free' approaches makes mutagenesis strategies in classical breeding indistinguishable from SDN-derived targeted genome modifications, even in regard to current regulatory rules. With the advent of new SDN technologies, much faster and more precise genome editing becomes available at reasonable cost, and potentially without requiring time-consuming deregulation of newly created phenotypes. This review will focus on classical mutagenesis breeding and the application of newly developed SDNs in order to emphasize similarities in the context of the regulatory situation for genetically modified crop plants.

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology.

Since the discovery that nucleases of the bacterial CRISPR (clustered regularly interspaced palindromic repeat)-associated (Cas) system can be used as easily programmable tools for genome engineering, their application massively transformed different areas of plant biology. In this review, we assess the current state of their use for crop breeding to incorporate attractive new agronomical traits into specific cultivars of various crop plants. This can be achieved by the use of Cas9/12 nucleases for double-strand break induction, resulting in mutations by non-homologous recombination. Strategies for performing such experiments - from the design of guide RNA to the use of different transformation technologies - are evaluated. Furthermore, we sum up recent developments regarding the use of nuclease-deficient Cas9/12 proteins, as DNA-binding moieties for targeting different kinds of enzyme activities to specific sites within the genome. Progress in base deamination, transcriptional induction and transcriptional repression, as well as in imaging in plants, is also discussed. As different Cas9/12 enzymes are at hand, the simultaneous application of various enzyme activities, to multiple genomic sites, is now in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology.

In planta gene targeting.

The development of designed site-specific endonucleases boosted the establishment of gene targeting (GT) techniques in a row of different species. However, the methods described in plants require a highly efficient transformation and regeneration procedure and, therefore, can be applied to very few species. Here, we describe a highly efficient GT system that is suitable for all transformable plants regardless of transformation efficiency. Efficient in planta GT was achieved in Arabidopsis thaliana by expression of a site-specific endonuclease that not only cuts within the target but also the chromosomal transgenic donor, leading to an excised targeting vector. Progeny clonal for the targeted allele could be obtained directly by harvesting seeds. Targeted events could be identified up to approximately once per 100 seeds depending on the target donor combination. Molecular analysis demonstrated that, in almost all events, homologous recombination occurred at both ends of the break. No ectopic integration of the GT vector was found.

The requirement for recombination factors differs considerably between different pathways of homologous double-strand break repair in somatic plant cells.

In recent years, multiple factors involved in DNA double-strand break (DSB) repair have been characterised in Arabidopsis thaliana. Using homologous sequences in somatic cells, DSBs are mainly repaired by two different pathways: synthesis-dependent strand annealing (SDSA) and single-strand annealing (SSA). By applying recombination substrates in which recombination is initiated by the induction of a site-specific DSB by the homing endonuclease I-SceI, we were able to characterise the involvement of different factors in both pathways. The nucleases MRE11 and COM1, both involved in DSB end processing, were not required for either SDSA or SSA in our assay system. Both SDSA and SSA were even more efficient without MRE11, in accordance with the fact that a loss of MRE11 might negatively affect the efficiency of non-homologous end joining. Loss of the classical recombinase RAD51 or its two paralogues RAD51C and XRCC3, as well as the SWI2/SNF2 remodelling factor RAD54, resulted in a drastic deficiency in SDSA but had hardly any influence on SSA, confirming that a strand exchange reaction is only required for SDSA. The helicase FANCM, which is postulated to be involved in the stabilisation of recombination intermediates, is surprisingly not only needed for SDSA but to a lesser extent also for SSA. Both SSA and SDSA were affected only weakly when the SMC6B protein, implicated in sister chromatid recombination, was absent, indicating that SSA and SDSA are in most cases intrachromatid recombination reactions.

RAD5A, RECQ4A, and MUS81 have specific functions in homologous recombination and define different pathways of DNA repair in Arabidopsis thaliana.

Complex DNA structures, such as double Holliday junctions and stalled replication forks, arise during DNA replication and DNA repair. Factors processing these intermediates include the endonuclease MUS81, helicases of the RecQ family, and the yeast SNF2 ATPase RAD5 and its Arabidopsis thaliana homolog RAD5A. By testing sensitivity of mutant plants to DNA-damaging agents, we defined the roles of these factors in Arabidopsis. rad5A recq4A and rad5A mus81 double mutants are more sensitive to cross-linking and methylating agents, showing that RAD5A is required for damage-induced DNA repair, independent of MUS81 and RECQ4A. The lethality of the recq4A mus81 double mutant indicates that MUS81 and RECQ4A also define parallel DNA repair pathways. The recq4A/mus81 lethality is suppressed by blocking homologous recombination (HR) through disruption of RAD51C, showing that RECQ4A and MUS81 are required for processing recombination-induced aberrant intermediates during replication. Thus, plants possess at least three different pathways to process DNA repair intermediates. We also examined HR-mediated double-strand break (DSB) repair using recombination substrates with inducible site-specific DSBs: MUS81 and RECQ4A are required for efficient synthesis-dependent strand annealing (SDSA) but only to a small extent for single-strand annealing (SSA). Interestingly, RAD5A plays a significant role in SDSA but not in SSA.

BRCA2 is a mediator of RAD51- and DMC1-facilitated homologous recombination in Arabidopsis thaliana.

• Mutations in the breast cancer susceptibility gene 2 (BRCA2) are correlated with hereditary breast cancer in humans. Studies have revealed that mammalian BRCA2 plays crucial roles in DNA repair. Therefore, we wished to define the role of the BRCA2 homologs in Arabidopsis in detail. • As Arabidopsis contains two functional BRCA2 homologs, an Atbrca2 double mutant was generated and analyzed with respect to hypersensitivity to genotoxic agents and recombination frequencies. Cytological studies addressing male and female meiosis were also conducted, and immunolocalization was performed in male meiotic prophase I. • The Atbrca2 double mutant showed hypersensitivity to the cross-linking agent mitomycin C and displayed a dramatic reduction in somatic homologous recombination frequency, especially after double-strand break induction. The loss of AtBRCA2 also led to severe defects in male meiosis and development of the female gametophyte and impeded proper localization of the synaptonemal complex protein AtZYP1 and the recombinases AtRAD51 and AtDMC1. • The results demonstrate that AtBRCA2 is important for both somatic and meiotic homologous recombination. We further show that AtBRCA2 is required for proper meiotic synapsis and mediates the recruitment of AtRAD51 and AtDMC1. Our results suggest that BRCA2 controls single-strand invasion steps during homologous recombination in plants.

The STRUCTURAL MAINTENANCE OF CHROMOSOMES 5/6 complex promotes sister chromatid alignment and homologous recombination after DNA damage in Arabidopsis thaliana.

Sister chromatids are often arranged as incompletely aligned entities in interphase nuclei of Arabidopsis thaliana. The STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC) 5/6 complex, together with cohesin, is involved in double-strand break (DSB) repair by sister chromatid recombination in yeasts and mammals. Here, we analyzed the function of genes in Arabidopsis. The wild-type allele of SMC5 is essential for seed development. Each of the two SMC6 homologs of Arabidopsis is required for efficient repair of DNA breakage via intermolecular homologous recombination in somatic cells. Alignment of sister chromatids is enhanced transiently after X-irradiation (and mitomycin C treatment) in wild-type nuclei. In the smc5/6 mutants, the x-ray-mediated increase in sister chromatid alignment is much lower and delayed. The reduced S phase-established cohesion caused by a knockout mutation in one of the alpha-kleisin genes, SYN1, also perturbed enhancement of sister chromatid alignment after irradiation, suggesting that the S phase-established cohesion is a prerequisite for correct DSB-dependent cohesion. The radiation-sensitive51 mutant, deficient in heteroduplex formation during DSB repair, showed wild-type frequencies of sister chromatid alignment after X-irradiation, implying that the irradiation-mediated increase in sister chromatid alignment is a prerequisite for, rather than a consequence of, DNA strand exchange between sister chromatids. Our results suggest that the SMC5/6 complex promotes sister chromatid cohesion after DNA breakage and facilitates homologous recombination between sister chromatids.

CRISPR-Cas9-mediated induction of heritable chromosomal translocations in Arabidopsis.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) technology has been applied in plant breeding mainly on genes for improving single or multiple traits1-4. Here we show that this technology can also be used to restructure plant chromosomes. Using the Cas9 nuclease from Staphylococcus aureus5, we were able to induce reciprocal translocations in the Mbp range between heterologous chromosomes in Arabidopsis thaliana. Of note, translocation frequency was about five times more efficient in the absence of the classical non-homologous end-joining pathway. Using egg-cell-specific expression of the Cas9 nuclease and consecutive bulk screening, we were able to isolate heritable events and establish lines homozygous for the translocation, reaching frequencies up to 2.5% for individual lines. Using molecular and cytological analysis, we confirmed that the chromosome-arm exchanges we obtained between chromosomes 1 and 2 and between chromosomes 1 and 5 of Arabidopsis were conservative and reciprocal. The induction of chromosomal translocations enables mimicking of genome evolution or modification of chromosomes in a directed manner, fixing or breaking genetic linkages between traits on different chromosomes. Controlled restructuring of plant genomes has the potential to transform plant breeding.

DNA Break Repair in Plants and Its Application for Genome Engineering.

Genome engineering is a biotechnological approach to precisely modify the genetic code of a given organism in order to change the context of an existing sequence or to create new genetic resources, e.g., for obtaining improved traits or performance. Efficient targeted genome alterations are mainly based on the induction of DNA double-strand breaks (DSBs) or adjacent single-strand breaks (SSBs). Naturally, all organisms continuously have to deal with DNA-damaging factors challenging the genetic integrity, and therefore a wide range of DNA repair mechanisms have evolved. A profound understanding of the different repair pathways is a prerequisite to control and enhance targeted gene modifications. DSB repair can take place by nonhomologous end joining (NHEJ) or homology-dependent repair (HDR). As the main outcome of NHEJ-mediated repair is accompanied by small insertions and deletions, it is applicable to specifically knock out genes or to rearrange linkage groups or whole chromosomes. The basic requirement for HDR is the presence of a homologous template; thus this process can be exploited for targeted integration of ectopic sequences into the plant genome. The development of different types of artificial site-specific nucleases allows for targeted DSB induction in the plant genome. Such synthetic nucleases have been used for both qualitatively studying DSB repair in vivo with respect to mechanistic differences and quantitatively in order to determine the role of key factors for NHEJ and HR, respectively. The conclusions drawn from these studies allow for a better understanding of genome evolution and help identifying synergistic or antagonistic genetic interactions while supporting biotechnological applications for transiently modifying the plant DNA repair machinery in favor of targeted genome engineering.

Two unlinked double-strand breaks can induce reciprocal exchanges in plant genomes via homologous recombination and nonhomologous end joining.

Using the rare-cutting endonuclease I-SceI we were able to demonstrate before that the repair of a single double-strand break (DSB) in a plant genome can be mutagenic due to insertions and deletions. However, during replication or due to irradiation several breaks might be induced simultaneously. To analyze the mutagenic potential of such a situation we established an experimental system in tobacco harboring two unlinked transgenes, each carrying an I-SceI site. After transient expression of I-SceI a kanamycin-resistance marker could be restored by joining two previously unlinked broken ends, either by homologous recombination (HR) or by nonhomologous end joining (NHEJ). Indeed, we were able to recover HR and NHEJ events with similar frequencies. Despite the fact that no selection was applied for joining the two other ends, the respective linkage could be detected in most cases tested, demonstrating that the respective exchanges were reciprocal. The frequencies obtained indicate that DSB-induced translocation is up to two orders of magnitude more frequent in somatic cells than ectopic gene conversion. Thus, DSB-induced reciprocal exchanges might play a significant role in plant genome evolution. The technique applied in this study may also be useful for the controlled exchange of unlinked sequences in plant genomes.