Gene Targeting of 5-Enolpyruvylshikimate-3-phosphate Synthase and Acetolactate Synthase for Transgene-Free Herbicide Resistance in Cassava

Weed competition on non-mechanized farms in Sub-Saharan Africa constrains production of cassava (Manihot esculenta) root, a staple food in this region. Availability of herbicide resistant elite hybrids could therefore increase the efficiency of cassava production for smallholder farms, but adoption of transgenic crops has not occurred in most of Africa. To assuage social perceptions against transgenic crops and provide durable weed control solutions for African cassava farmers, we are targeting the acetolactate synthase (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) genes to introduce amino acid substitutions that are likely to confer resistance to all major classes of ALS inhibitors and to glyphosate, respectively. In plant cells, introduction of a DNA double strand break (DSB) at a target site can significantly stimulate the low frequency of homologous recombination. Delivery of the repair template on a geminivirus replicon (GVR) was recently shown to provide a further 10- to 100-fold increase of homology-directed repair (HDR) compared to a non-replicating control, presumably due in part to amplification of the template sequence during GVR replication. Thus, we have built and tested transcription activator-like effector nucleases and RNA-guided Cas9 nucleases specific for cassava ALS or EPSPS target sites and applied them to cassava cells simultaneously with a corresponding GVR-borne repair template. The first demonstration of gene targeting was initiated by insertion of a reporter-marker fusion downstream of the cassava genes, allowing event observation and selection on kanamycin. Subsequent work will use repair templates containing only ALS and EPSPS sequence with amino acid substitutions conferring herbicide resistance.