EAGER: A Transient System for Cassava Genome Editing

Cassava (Manihot esculenta Crantz) is the second most important crop in Africa after maize. East Africa is the world's largest producer of cassava, accounting for ~60% of world production, where it is often grown on marginal lands. Millions of people depend on cassava as their major source of calories, and it plays a key role in food security for the developing world. It is also an important source of income for small farmers, who are often women in Africa. Many different cassava cultivars that are adapted to local environmental conditions and have unique tuber properties are grown across Africa. Farmers often prefer local varieties instead of 'improved' cultivars that are less susceptible to disease and pests, which greatly limit cassava production in Africa. Thus, it is important to develop genetic approaches to improve local varieties as well as elite cultivars that are part of national breeding programs. This EAGER project will develop an innovative, rapid strategy for modifying the cassava genome that leaves no 'footprint' except for the desired mutation. This strategy has many potential applications and would complement conventional breeding and transgenic approaches used today for cassava improvement. In addition, since cassava geminiviruses are members of the begomovirus genus that includes nearly 300 viral species, the information and resources generated could also provide new insight into how other begomoviruses can be adapted as viral vectors for transient gene editing systems, potentially extending the technology to many important crop species in addition to cassava.

This project will develop a new approach using viral vectors to express targeted nucleases to introduce site-specific modifications into the cassava genome. The system will use a viral vector based on a cassava geminivirus to express a meganuclease designed to introduce mutations into the cassava gene encoding phytoene desaturase (PDS), the first enzyme in the carotenoid biosynthetic pathway. The bleached phenotype of a PDS mutant will be used to screen for gene knockouts caused by the transient gene editing system and establish its efficacy. The impact of several parameters, e.g. the inoculation method, inoculation site, virus movement and cassava sequences that enhance infection, on the efficiency of the system will be tested. Experiments will also characterize progeny plants for the presence of mutant PDS sequences as well as for viral and meganuclease sequences. These studies will indicate if vegetative propagation of the inoculated cassava increases the efficiency of detecting a site-specific mutation and if progeny plants do not retain viral or meganuclease DNA.