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

Journal Article · Thu Jan 18 00:00:00 EST 2024 · Plant Physiology (Bethesda)

DOI:https://doi.org/10.1093/plphys/kiae015· OSTI ID:2329550

Liu, Degao ; Myers, Erik A. ; Xuan, Shuya ; Prichard, Lynn E. ; Donahue, Lilee I. ; Ellison, Evan E. ; Starker, Colby G. ; Voytas, Daniel F.

Abstract CRISPR/Cas9-mediated mutagenesis typically results in short insertion/deletion 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 multinucleotide deletions in both protein-coding genes and noncoding DNA regulatory elements. VirTREX2-HLDel uses RNA viruses to deliver both the 3 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.

• Sponsoring Organization: USDOE
• Grant/Contract Number: SC-0018277; SC-0023160
• OSTI ID: 2329550
• Alternate ID(s): OSTI ID: 2283101
• Journal Information: Plant Physiology (Bethesda), Journal Name: Plant Physiology (Bethesda) Vol. 194 Journal Issue: 4; ISSN 0032-0889
• Publisher: Oxford University Press
• Country of Publication: United States
• Language: English