meiTALENs: Directing crossover recombination with meiotic TAL nucleases

There is a critical need to improve crop yields to provide food security for the growing human population. Harnessing the genetic variation present in crops and their wild relatives is critical for crop improvement. Most crops have passed through domestication bottlenecks and so contain a fraction of wild genetic diversity. Crossing can therefore reintroduce useful variation from wild strains, for example in disease and stress resistance and increase yield. This process is dependent on recombination and independent chromosome segregation that occurs during meiosis. Meiotic recombination can produce crossovers between chromosomes and generation of novel combinations of genetic variation. Where crossovers occur in the genome is non-random, which can impose a limitation on breeding. For example, many important crops such as maize, wheat, barley and tomato have very skewed crossover patterns, with the majority of events occurring towards the end of the chromosomes. Therefore, useful variation located in the centre of crop chromosomes can be difficult for breeders to fully utilise. In the proposed work we will develop synthetic tools that will allow scientists and breeders to direct crossover recombination to specific sites in the genome. This will be done by fusing programmable DNA binding domains to nucleases and expressing them during the meiotic sexual cell division. This will drive breaks in the DNA at target sites, which will then be repaired as crossovers via the specialized machinery present during meiosis. We will perform this work as a proof-of-principle in the model species Arabidopsis thaliana, which has extensive genetic and genomic resources that will ensure rapid progress and demonstration of functionality. For example, we have developed fluorescent systems that allow us to score 100,000s of meiosis in 10-15 minutes. As the recombination machinery is highly conserved between plants our understanding will be directly applicable to crop species. This work will take advantage of cutting-edge synthetic biology to manipulate the core process of meiotic recombination, with direct impact for crop improvement.

Meiotic recombination shapes genetic variation and is critically important during crop breeding. During meiosis homologous chromosomes undergo reciprocal exchange, termed crossover (CO). We have shown that the majority of recombination is concentrated in hotspots, with 80% of COs occurring in 26% of the sequence. A major determinant of hotspot location is chromatin and hotspots do not share an underlying sequence motif. Therefore, we propose to take a synthetic approach to direct Arabidopsis COs to specific DNA sequences. We will fuse TAL DNA binding domains to nucleases and express them during meiosis. We will transform into wild type and spo11 mutants, which lack endogenous meiotic DNA double strand breaks. As chromosomes fail to pair in spo11, this causes a failure in balanced chromosome segregation and sterility. Therefore, we will screen for meiTALENs that increase spo11 fertility, which will indicate formation of COs. Equally, we will screen for meiTALENs that decrease fertility in wild type, which will indicate formation of excessive DSBs. We have designed TAL arrays to recognize 10s, 100s and 1000s of sites throughout the genome. We have also designed arrays to the telomeric and centromeric repeats, which are highly non-randomly distributed along chromosomes. meiTALENs will be combined with fluorescent segregation systems that allow 100,000s meioses per individual to be scored using flow cytometry. We will perform fine-scale genetic mapping to demonstrate that meiTALEN COs occur at the predicted target sequences. Finally, we will combine meiTALENs with mutants known to alter crossover recombination in order to test mechanistic hypotheses. This will include chromatin (met1 and arp6) and recombination (fancm) mutants. This work will be performed as a proof-of-principle in Arabidopsis where we have the strategic advantages of rapid generation time, detailed genomic and epigenomic annotation, recombination maps and crossover measurement assays.

Breeding of natural genetic variation remains a critical tool for crop improvement. The majority of agricultural crops contain a fraction of the genetic diversity present in their wild progenitors. Reintroduction of variation in disease resistance and stress tolerance will allow higher yielding, more sustainable crops to be developed. One limitation to breeding is the skewed distribution of crossover events in crop species, including wheat, barley, maize and tomato. Tools to control crossover distributions will have general utility in the context of crop breeding. Promotion of high levels of crossover throughout the genome, or in specific locations, would allow breeders to generate populations with novel combinations of traits. Equally, it will also be useful to suppress crossovers to maintain linkage between useful combinations of genes, for example disease resistance genes. The meiTALEN strategy therefore will have direct utility in crop genomes. Currently, crop genomes are either incomplete or transformation is not possible. Therefore, we propose to define meiTALEN functionality in Arabidopsis, and translate this mechanism as crop genetic resources continue to develop. To ensure translation of meiTALEN technology into crops we have developed strategic links with plant breeding industry. Specifically, via a BBSRC-CASE studentship with Syngenta and an industrial studentship, currently under negotiation, with Rijk Zwaan. Through these links we will explore application of meiTALENs in wheat, tomato and carrot, where there is demonstrated need to modify COs during breeding, to maximize use of available genetic variation.