Generation of inheritable and 'transgene clean' targeted genome-modified rice in later generations using the CRISPR/Cas9 system.

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. [ABSTRACT FROM AUTHOR]