Project Proposal: Channeling targeted DNA double strand breaks into alternative repair pathways

The Idea

We have transformed Arabidopsis with TAL-nucleases expressed from meiotic promoters in order to direct recombination to sequences of choice. This approach has the potential to unlock non-recombining regions of plant genomes and accelerate breeding. Our preliminary data indicates that DNA double strand breaks generated by the FokI nuclease present in our TALEN constructs are not entering the inter-homolog repair pathway that can lead to crossover recombination. Instead they are being repaired via non-homologous end joining (NHEJ), causing deletions. We propose to repeat this approach in NHEJ mutant backgrounds (ku70 xrcc1) in order to channel TALEN-induced DSBs into inter-homolog repair.

Who We Are

Dr Ian Henderson, Dr Natasha Yelina, Patrick Diaz (Department, of Plant Sciences, University of Cambridge)

Dr Sebastian Schornack (The Sainsbury Laboratory, University of Cambridge)

Meiogenix (Paris)

Implementation

Channeling targeted DNA double strand breaks into alternative repair pathways

Aims: Use mutants defective in DNA repair pathways to channel targeted DNA double strand breaks into different repair pathways.

Methods: We have established GoldenGate TALEN synthesis in the laboratory and generated TAL-FokI pairs expressed from meiotic promoters (DMC1, SPO11). These constructs will be transformed into wild type and NHEJ mutants and transform and T1 progeny analysed phenotypically and via Illumina sequencing of their DNA. Mutations will be identified using established pipelines for SNP and indeed calling. All lines will be in the Col TAIR10 reference background.

Outcomes: A deeper understanding of recombination pathways in Arabidopsis and how they can be manipulated in ways beneficial to crop breeding.

Who will be involved: The TALEN work in our lab is being driven by Dr Natasha Yelina and a graduate student Patrick Diaz. Bioinformatics and computational analysis will be performed by Dr Ian Henderson and Dr Xiaoyu Zhao. We also actively collaborate with a company Meiogenix who are developing parallel targeted recombination technology.

Benefits and outcomes

Meiotic recombination distributions are a major limitation to crop breeding. For example, much of the large, repetitive wheat genome is non-recombining, which slows the generation of useful recombinants. This technology will allow meiotic recombination to be targeted to regions of choice and accelerate crop breeding.

Budget

Our TALEN work is funded by a BBSRC grant, which supports the synthesis and transformation of meiotically expressed TALENs. Our recent data support the unexpected outcome that TALEN-induced DSBs are being channelled into a non-crossover repair pathway. To confirm this we request  £4000 to fund 2 lanes of mid-output Nextseq Illumina sequencing (~£2000 per lane). This data will allow us to sequence TALEN progeny in wild type and recombination mutant backgrounds to directly monitor genetic change caused by our targeted recombination systems. All necessary bioinformatics and experimental techniques are established in the lab.