Heritable, multinucleotide deletions in plants using viral delivery of a repair exonuclease and guide RNAs.

CRISPR/Cas9-mediated mutagenesis typically results in short insertion/deletion (indel) mutations, which are often too small to disrupt the function of cis-acting regulatory elements. Here, we describe a highly efficient in planta gene editing approach, called VirTREX2-HLDel, that achieves heritable multi-nucleotide deletions in both protein-coding genes and non-coding DNA regulatory elements. VirTREX2-HLDel uses RNA viruses to deliver both the three prime repair exonuclease 2 (TREX2) and single guide RNAs. Our method enables recovery of multiplexed heritable deletions and increases the heritable gene editing frequency at poorly edited sites. We identified functional conservation and divergence of MICRORNA164 (miR164) in Nicotiana benthamiana and tomato (Solanum lycopersicum) using VirTREX2-HLDel and observed previously uncharacterized phenotypes in plants with large deletions at this locus. Our viral delivery method reduces the need for tissue culture and will accelerate the understanding of protein-coding and regulatory regions in plants.