**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**

**Qingsong Gao 1,**†**, Gang Li 2,**†**, Hui Sun 1, Ming Xu 1, Huanhuan Wang 1, Jianhui Ji 1, Di Wang 2, Caiyong Yuan 2,\* and Xiangxiang Zhao 1,\***


Received: 30 November 2019; Accepted: 22 January 2020; Published: 26 January 2020

**Abstract:** The *FW2.2*-like (*FWL*) 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 *FWL* genes using the CRISPR/Cas9 system delivered by *Agrobacterium*-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 *OsFWL4* gene mutants are significantly greater than those of the wild type. Flag leaves of *OsFWL4* 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 *OsFWL1* 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 *Agrobacterium*-mediated CRISPR/Cas9 system and that the *OsFWL4* gene is a negative regulator of tiller number and plant yield.

**Keywords:** *FW2.2*-like gene; tiller number; grain yield; rice; CRISPR/Cas9; genome editing; off-target effect
