a A schematic representation of the gene drive that is inserted into the second exon of the CRYPTOCHROME 1 (CRY1) gene on Chromosome IV using CRISPR/Cas9-mediated homology-directed repair (HDR). The gene drive element flanked with homology arms is released by the Cas9/gRNA2 complex, which is produced by the Cas9 and gRNA units on the gene drive. The gene drive element has several components: a Cas9-expressing unit, two gRNA units (one targeted CRY1 and the other was used to release the HDR template from the plasmid). The GFP gene is designed to fuse with part of CRY1 in frame, providing a visual marker for precise insertion of the gene drive element. The mCherry is another marker for selecting transgenic events. The Cas9 target sequence in CRY1 with the CCC (underlined) protospacer adjacent motif (PAM) is shown. The HDR events can be identified using PCR-based methods. Both P1 (CRY1-LA-GT-5P) and P4 (CRY1-RA-GT-3P) were located outside of the homology arms. b Conversion of heterozygous F1 plants into homozygous plants by the gene drive. The gene drive unit shown in a is shown in red here. When the homozygous gene drive line (♀), which was generated in the Columbia (Col) background (in black), was crossed to Landsberg (Ler, in blue), the F1 plants should contain just one copy of the gene drive at the CRY1 locus. When the gene drive functions as designed, the Cas9/gRNA complex cuts CRY1 in the Ler chromosome, and the gene drive element in the Col chromosome is copied into the Ler CRY1 locus, resulting in homozygous F1 plants.