The CRISPR/Cas9 system is becoming an important genome editing tool for crop breeding. Although it has been demonstrated that target mutations can be transmitted to the next generation, their inheritance pattern has not yet been fully elucidated. Here, we describe the CRISPR/Cas9-mediated genome editing of four different rice genes with the help of online target-design tools. High-frequency mutagenesis and a large percentage of putative biallelic mutations were observed in T₀ generations. Nonetheless, our results also indicate that the progeny genotypes of biallelic T₀ lines are frequently difficult to predict and that the transmission of mutations largely does not conform to classical genetic laws, which suggests that the mutations in T₀ transgenic rice are mainly somatic mutations. Next, we followed the inheritance pattern of T₁ plants. Regardless of the presence of the CRISPR/Cas9 transgene, the mutations in T₁ lines were stably transmitted to later generations, indicating a standard germline transmission pattern. Off-target effects were also evaluated and our results indicate that with careful target selection, off-target mutations are rare in CRISPR/Cas9-mediated rice gene editing. Taken together, our results indicate the promising production of inheritable and “transgene clean” targeted genome-modified rice in the T₁ generation using the CRISPR/Cas9 system.

Background: The type II clustered, regularly interspaced, short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) system is a novel molecular tool for site-specific genome modification. The CRISPR-Cas9 system was recently introduced into plants by transient or stable transformation. Findings: Here, we report gene targeting in rice via the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system. Three 20-nt CRISPR RNAs were designed to pair with diverse sites followed by the protospacer adjacent motif (PAM) of the rice herbicide resistance gene BEL. After integrating the single-guide RNA (sgRNA) and Cas9 cassette in a single binary vector, transgenic rice plants harboring sgRNA:Cas9 were generated by A. tumefaciens-mediated stable transformation. By analyzing the targeting site on the genome of corresponding transgenic plants, the mutations were determined. The mutagenesis efficiency was varied from ~2% to ~16%. Furthermore, phenotypic analysis revealed that the biallelic mutated transgenic plant was sensitive to bentazon. Conclusions: Our results indicate that the agricultural trait could be purposely modified by sgRNA:Cas9-induced gene targeting. CRISPR-Cas9 system could be exploited as a powerful tool for trait improvements in crop breeding.

The E. coli phosphomannose isomerase (EcPMI) gene is widely used as a selectable marker gene (SMG) in mannose (Man) selection-based plant transformation. Although some plant species exhibit significant PMI activity and active PMIs were even identified in Man-sensitive plants, whether plant PMIs can be used as SMGs remains unclear. In this study, we isolated four novel PMI genes from Chlorella variabilis and Oryza sativa. Their isoenzymatic activities were examined in vitro and compared with that of EcPMI. The active plant PMIs were separately constructed into binary vectors as SMGs and then transformed into rice via Agrobacterium. In both Indica and Japonica subspecies, our results indicated that the plant PMIs could select and produce transgenic plants in a pattern similar to that of EcPMI. The transgenic plants exhibited an accumulation of plant PMI transcripts and enhancement of the in vivo PMI activity. Furthermore, a gene of interest was successfully transformed into rice using the plant PMIs as SMGs. Thus, novel SMGs for Man selection were isolated from plants and our analysis suggested that PMIs encoding active enzymes might be common in plants and could potentially be used as appropriate genetic elements in cisgenesis engineering.

Summary: Using promoters expressed in nonendosperm tissues to activate target genes in specific plant tissues or organs with very limited expression in the endosperm is an attractive approach in crop transgenic engineering. In this article, five putative nonendosperm tissue‐expressed promoters were cloned from the rice genome and designated P_OsNETE1, P_OsNETE2, P_OsNETE3, P_OsNETE4 and P_OsNETE5. By qualitatively and quantitatively examining GUSplus reporter gene expression in transgenic rice plants, P_OsNETE1‐P_OsNETE5 were all found to be active in the roots, leaves, stems, sheaths and panicles but not in the endosperm of plants at different developmental stages. In addition, P_OsNETE2, P_OsNETE4 and P_OsNETE5 were also inactive in rice embryos. Among these promoters, P_OsNETE4 and P_OsNETE5 exhibited higher activities in all of the tested tissues, and their activities in stems, leaves, roots and sheaths were higher than or comparable to those of the rice Actin1 promoter. We also progressively monitored the activities of P_OsNETE1‐P_OsNETE5 in two generations of single‐copy lines and found that these promoters were stably expressed between generations. Transgenic rice was produced using P_OsNETE4 and P_OsNETE5 to drive a modified Bt gene, mCry1Ab. Bt protein expressed in the tested plants ranged from 1769.4 to 4428.8 ng/g fresh leaves, whereas Bt protein was barely detected in the endosperm. Overall, our study identified five novel nonendosperm tissue‐expressed promoters that might be suitable for rice genetic engineering and might reduce potential social concern regarding the safety of GMO crops.