Knockout of the gene in rice via targeted mutagenesis using the CRISPR/Cas9 system

Saika, Hiroaki*; Mikami, Masafumi*; Endo, Masaki*; Sakamoto, Ayako; Toki, Seiichi*

Arabidopsis thaliana cells, in which DNA double-strand breaks (DSBs) have occurred, select either DNA repair, cell death or endocycle. Avoidance of entry into endocycle even under genotoxic conditions, rice (Oryza sativa L.) is more suitable than Arabidopsis for the study of DSBs repair in plants. Nijmegen breakage syndrome 1 (NBS1) protein consists of the Mre11/Rad50/NBS1 (MRN) complex plays an important role in response to DNA damage in mammal. Although rice has a homologue of NBS1, OsNBS1, its function remains unclear. Here, we attempted to knockout the OsNBS1 gene using the recently developed clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system. Rice (cv. Nipponbare) cells were transformed via Agrobacterium and obtained 72 lines of cali. Cleaved amplified polymorphic sequences (CAPS) analysis detected mutations in 58 lines. Biallelic mutations in theOsNBS1 gene were induced in 24 lines. Sequence analyses of PCR products in #2 and #4 detected not only small mutations but also large modifications at the expected cleavage site. The large insertions involved one or two fragments that seem to be copied from sequences around the cleavage site. These mutations are thought to be repaired by a synthesis-dependent strand annealing (SDSA)-like system as proposed in a previous report in barley.