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.
Highlights
ResultsTwo target sites were designed in the coding region of each of the eight rice FWL genes for clustered regularly interspaced short palindromic repeats (CRISPR)/Cas gene editing (Table 1)
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
Two target sites were designed in the coding region of each of the eight rice FWL genes for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing (Table 1)
Summary
Two target sites were designed in the coding region of each of the eight rice FWL genes for CRISPR/Cas gene editing (Table 1). To test whether failed editing of these plants was caused by a lack of the CRISPR/Cas construct, the presence of hygromycin phosphotransferase (HPT), sgRNA, and Cas transgenes in these 41 plants was examined. Two plants did not contain HPT, sgRNA, and Cas sequences (Table S2), which suggests that these plants escaped hygromycin selection. When unmutated T0 plants without the complete sgRNA/Cas construct were excluded, all targets except Osfwl1a, Osfwl4a, and Osfwl6a had a mutation rate of 100% (Table 2 and Table S2). Examined the T0 plant of this line for the presence of a T-DNA fragment and found that it lacked sgRNA and Cas sequences. This indicates that mutations of this line were generated by transient CRISPR/Cas expression
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