However, mutations occurring randomly under natural conditions have provided very limited genetic resources for yield increases. In this study, potentially yield-increasing alleles of two genes closely associated with yield were edited artificially. The recently developed CRISPR/Cas9 system was used to edit two yield genes: Grain number 1a (Gn1a) and DENSE AND ERECT PANICLE1 (DEP1). Several mutants were identified by a target sequence analysis. Phenotypic analysis confirmed one mutant allele of Gn1a and three of DEP1 conferring yield superior to that conferred by other natural high-yield alleles. Our results demonstrate that favorable alleles of the Gnla and DEP1 genes, which are considered key factors in rice yield increases, could be developed by artificial mutagenesis using genome editing technology.
Figure 1: Flow diagram of artificial mutagenesis.
A: pCAMBIA1300-OsU3-Cas9 binary vector, where the hSpCas9 cassette is driven by the 2× 35S promoterand sgRNA (simple guide RNA) is controlled by the rice OsU3 promoter. The target region contains two Aar I enzyme sites, and HYG (hygromycin) was used as a resistance selection marker.
C: Albino phenotype caused by OsPDS mutation.
D: Gel image of the target gene after Pst I digestion. M, marker. 1–9, mutation lines of OsPDS. wt, wild type. The mutation in CTGCAG (Pst I site) cannot be cut by the Pst I enzyme.
E: Sequence of some mutation lines aligned with wild-type OsPDS gene sequence. −11 and +1 represent an 11-bp deletion and a 1-bp insertion, respectively.