Targeted Mutagenesis of the Rice FW 2.2-Like Gene Family Using the CRISPR/Cas9 System Reveals OsFWL4 as a Regulator of Tiller Number and Plant Yield in Rice

Abstract

The <i>FW2.2</i>-like (<i>FWL</i>) genes encode cysteine-rich proteins with a placenta-specific 8 domain. They play roles in cell division and organ size control, response to rhizobium infection, and metal ion homeostasis in plants. Here, we target eight rice <i>FWL</i> genes using the CRISPR/Cas9 system delivered by <i>Agrobacterium</i>-mediated transformation. We successfully generate transgenic T₀ lines for 15 of the 16 targets. The targeted mutations are detected in the T₀ lines of all 15 targets and the average mutation rate is found to be 81.6%. Transfer DNA (T-DNA) truncation is a major reason for the failure of mutagenesis in T₀ plants. T-DNA segregation analysis reveals that the T-DNA inserts in transgenic plants can be easily eliminated in the T₁ generation. Of the 30 putative off-target sites examined, unintended mutations are detected in 13 sites. Phenotypic analysis reveals that tiller number and plant yield of <i>OsFWL4</i> gene mutants are significantly greater than those of the wild type. Flag leaves of <i>OsFWL4</i> gene mutants are wider than those of the wild type. The increase in leaf width of the mutants is caused by an increase in cell number. Additionally, grain length of <i>OsFWL1</i> gene mutants is higher than that of the wild type. Our results suggest that transgene-free rice plants with targeted mutations can be produced in the T₁ generation using the <i>Agrobacterium</i>-mediated CRISPR/Cas9 system and that the <i>OsFWL4</i> gene is a negative regulator of tiller number and plant yield.