Transgene- and tissue culture-free heritable genome editing using RNA virus-based delivery in wheat

Abstract

CRISPR-Cas genome editing technology is a cutting-edge strategy for crop breeding. However, the delivery of genome-editing reagents remains to be a technological bottleneck in monocot plants¹. Here we engineered barley yellow striate mosaic virus (BYSMV) into a negative-strand RNA virus-based vector system² for delivery of both Cas9 and single guide RNA to achieve heritable gene editing in different wheat cultivars. We found that fusion of a mobile transfer RNA sequence³ to the Cas9 messenger RNA and single guide RNAs could deliver them into the growth points of axillary meristems to achieve gene editing before tiller generation. The resulting nascent tillers contained simultaneous mutations in the three homoeoalleles. Moreover, the progeny seedlings are virus-free and harbour bi-allelic or homozygous mutations. Given BYSMV infects 26 monocot species⁴, the BYSMV delivery system could have wide applicability for achieving highly efficient, non-transgenic and less genotype-dependent heritable genome editing, thereby facilitating genomic studies and crops breeding.