Loss-of-function mutation in the polyamine transporter gene OsLAT5 as a selectable marker for genome editing

Abstract

Genome editing by TALENs, CRISPR/Cas, base or prime editing have become routine tools. During stable plant transformation, the gene coding for the editing enzyme, e.g., Cas9, the guide RNAs (gRNAs), alongside a selectable marker are integrated into the nuclear genome. Identification of successful transformants relies on selectable or screenable markers, typically genes providing resistance to antibiotics or herbicides. Selectable markers use a substantial portion of the T-DNA, hence reducing transfer efficiency by limiting the effective number of TALENs or guide/pegRNAs that can be used in parallel. Moreover, marker genes are frequently subject to gene silencing. Here, we generated loss-of-function mutations in PUT/LAT-type polyamine transporter family genes to confer resistance to the phytotoxin methylviologen (MV) as a method for selection. As a proof of concept, CRISPR/Cas9 vectors with gRNAs were constructed to target three close homologs OsLAT1, OsLAT5, and OsLAT7. We show that loss of OsLAT5 (also known as OsPUT3 or OsPAR1) function was sufficient to confer resistance to MV in rice seeds, seedlings and calli, validating the editing approach of OsLAT5 to obtain a selectable marker. We discuss the potential of incorporating a gRNA cassette (for OsLAT5) as a selectable marker and a reporter for successful genome editing for optimizing editing protocols.