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

Abstract

Abstract Genome editing by TALENs and CRISPR/Cas has become routine tools. During stable plant transformation, genes coding for editing enzymes, e.g., Cas9, guide RNAs (gRNA), and selectable markers are integrated into the nuclear genome. Identification of successful transformants relies on selectable or screenable markers, typically genes providing resistance to herbicides or antibiotics. 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. 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 methylviologen (MV). As proof of concept, CRISPR/Cas9 constructs with gRNAs were constructed to target three close homologs OsLAT1, OsLAT5, and OsLAT7. 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 use of a gRNA cassette (OsLAT5) as selectable marker and reporter for successful genome editing for optimizing editing protocols.