A review-based guide to selecting an appropriate strategy for transgene-free genome editing in plants

The field of plant genetic engineering has advanced considerably since the development of the first transgenic crop plants, and even though transgenic varieties of various crops are now commercially available, consumer reluctance and environmental concerns over transgene escape pose limits on their expansion. Gene editing offers the opportunity to selectively and precisely alter DNA sequences without reliance on stably integrated transgenes, assuaging both concerns. Considerable progress has been made with the advent of programmable nucleases such as Cas9 and its derivatives. The transgenes encoding the gene editing components can be expressed transiently without integration in the plant's genome or removed from the genome via outcrossing or haploid induction to result in gene-edited plants that are no longer considered transgenic. Nonetheless, gene editing systems remain challenging to implement in some plant species, notably those that have long generation times or are vegetatively propagated, precluding the removal of editing tools through crosses. In these cases, a transgene-free method for introduction of all components needed to enable gene editing will bring the promise of modern breeding tools within reach. Transgene-free genome editing can be accomplished through the delivery of editing components as DNA, RNA, or ribonucleoproteins with delivery via Agrobacterium, microprojectiles, nanocarriers, DNA and RNA viruses, and grafting. Between technical constraints and scientific goals, it can be challenging to determine an appropriate transgene-free genome editing method. Following a review of the literature, a decision tree is provided to help select an appropriate method for a given plant species and to show how emerging tools, including smaller editors, can advance the field.