Inactivation of retrotransposon Tos17 Chr.7 in rice cultivar Nipponbare through CRISPR/Cas9-mediated gene editing.

Retrotransposons are mobile genetic elements capable of transposition via reverse transcription of RNA intermediates. Rice cultivar Nipponbare contains two nearly identical genomic copies of Tos17, an endogenous copia-like LTR retrotransposon, on chromosomes 7 (Tos17 Chr.7) and 10 (Tos17 Chr.10), respectively. Previous studies demonstrated that only Tos17 Chr.7 is active in transposition during tissue culture. Tos17 Chr.7 has been extensively used for insertional mutagenesis as a tool for functional analysis of rice genes. However, Tos17 Chr.7 transposition might generate somaclonal mutagenesis with undesirable traits during rice transformation, which would affect the evaluation or application of transgenes. In this study, we generated a Tos17 Chr.7 knockout mutant D873 by using CRISPR/Cas9 gene editing system. The gene-edited allele of Tos17 Chr.7 in D873, designated as Tos17 D873, has an 873-bp DNA deletion in the pol gene of Tos17 Chr.7, which caused the deletion of the GAG-pre-integrase domain and the integrase core domain. Although the transcription of Tos17 D873 was activated in D873 calli, no transposition of Tos17 D873 was detected in the regenerated D873 plants. The results demonstrate that the GAG-pre-integrase domain and the integrase core domain are essential for Tos17 Chr.7 transposition and the deletion of the two domains could be not complemented by other LTR retrotransposons in rice genome. As the Tos17 Chr.7-derived somaclonal mutagenesis is blocked in the D873 plants, the generation of the Tos17 D873 allele will be helpful in production of transgenic rice plants for gene function study and genetic engineering. Similar approach can be used to inactivate other retrotransposons in crop breeding.