**1. Introduction**

*fw 2.2* is a major quantitative trait locus that regulates fruit size and weight in tomato [1,2]. The underlying gene *FW2.2* regulates fruit size by negatively regulating cell division [2,3]. Homolog identification and sequence analysis have revealed that FW2.2 belongs to a large eukaryotic family of cysteine-rich proteins containing a featured placenta-specific 8 domain [4,5]. *FW2.2*-like (*FWL*) genes have been characterized in various plant species and are reported to play important roles in diverse biological processes, such as cell number and organ size control [4,6–9], nodulation [5,10], and metal ion homeostasis [11–20]. The rice *FWL* gene family contains eight members [9]. Among them, the *OsFWL3* gene is reported to negatively affect grain length and weight by regulating cell division in the glume [9]. However, the *OsFWL4* gene has been reported to affect cadmium (Cd) resistance upon

expression in yeast [15]. RNA interference-mediated knockdown of *OsFWL4* has been found to reduce translocation of Cd from the roots to shoots in rice seedlings. More recently, a change in the expression of *OsFWL1* and *OsFWL2* induced by their overexpression or RNA interference has been found to affect Cd tolerance and accumulation in rice [20]. Interestingly, the *OsFWL5*/*PCR1* gene, which affects Cd and Zn tolerance when expressed in yeast cells, has been reported to regulate metal ion homeostasis and grain size and weight in rice [13,14]. However, whether other Cd-responsive rice *FWL* genes also play a role in plant and organ development in rice remains unknown.

Generating mutants with intended mutations is crucial for functional analysis of plant genes. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a powerful tool for genome editing in various organisms, including plants. This system induces DNA double-strand breaks at given genomic sites, which are subsequently repaired by either non-homologous end joining or homologous recombination pathways in the cells [21]. Non-homologous end joining is error-prone and can act throughout the cell cycle. It is therefore commonly utilized to disrupt genes by creating random insertions or deletions (indels) at target sites [22]. In the presence of a homologous DNA template, a double-strand break can be repaired by homologous recombination, leading to target gene replacement or insertion. The precise cleavage of the target DNA using the CRISPR/Cas9 system requires two components, namely, Cas9 nuclease harboring HNH and RuvC endonuclease domains for cleaving and an engineered single-guide RNA (sgRNA) for directing Cas9 to the target site [23]. A prerequisite for binding and cleavage of the target DNA is the presence of a trinucleotide protospacer adjacent motif (PAM) immediately after the target DNA [24]. Sequence specificity can be achieved by changing a 20-nucleotide "guide sequence" in the sgRNA. As this system does not require protein engineering, the nuclease can be easily reprogrammed. With the development of highly efficient CRISPR/Cas9 systems, stable homozygous mutants can be obtained within a single generation in many plants [25–28].

*Agrobacterium*-mediated transfer DNA (T-DNA) transformation is commonly used for delivering CRISPR/Cas9 DNA into rice cells. In this study, two target sites are designed for each of the eight rice *FWL* genes for gene editing using the *Agrobacterium*-mediated CRISPR/Cas9 system. We generate transgenic T0 lines from 15 out of 16 constructs and detect targeted mutations in all T0 lines. Gene editing efficiency, T-DNA segregation patterns, and off-target effects are analyzed. The phenotypes of homozygous and transgene-free mutants with no detected off-target mutations of the *OsFWL1* and *OsFWL4* genes are then examined. Our results suggest that OsFWL4 is a negative regulator of tiller number and plant yield in rice and that OsFWL1 plays a role in modulating rice grain length.
