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Segregation of genetic chimeras generated by CRISPR/Cas9 system in rice

Lee, Sangyool, Kim, Ju-Kon, Choi, Yang Do, Jang, Geupil
Plant biotechnology reports 2019 v.13 no.1 pp. 35-42
Oryza sativa, adaptive immunity, bacteria, chimerism, gene editing, homozygosity, mutants, mutation, plasmids, rice, transgenic plants
CRISPR/Cas9 system based on the adaptive immune system of bacteria is an emerging powerful technique of genome editing. Previously, we showed that transgenic rice transformed with a recombinant CRISPR/Cas9-OsJAZ9 binary plasmid display genetic chimerism in T₀ generation, and the genetic chimerism includes homozygous and heterogeneous mutation of the target gene, OsJAZ9. The homozygous mutation of the T₀ lines was stably transferred into the next generation, and all T₁ plants of the T₀ homozygous mutants were homozygous mutants. However, the fate of T₁ plants with heterogeneous mutation remains poorly understood. In this study, we characterized target gene mutation in the progenies of the T₀ lines with heterogeneous mutation to understand fate of the T₀ lines with heterogeneous mutation. We found that some T₀ lines such as lines 6 and 21 produce the T₁ plants carrying homozygous mutation of OsJAZ9 through genetic segregation, while other T₀ lines such as lines 3, 4 and 15 did not produce homozygous mutants in T₁ generation unlike lines 6 and 21. These indicated that 40% of T₀ lines with heterogeneous mutation produce the plants with homozygous mutation in T₁ generation, suggesting that every T₀ heterogeneous line does not produce the progenies with homozygous mutation through genetic segregation. However, because the portion of T₀ lines with heterogeneous mutation (61%) is much bigger than that of T₀ lines with homozygous mutation (8%) in T₀ pool, these results propose that T₀ heterogeneous mutants are useful to obtain CRISPR/Cas9-mediated homozygous mutants.