Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus Using CRISPR-Cas9
November 23, 2016| |
The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes. Longxiang Wang, from Huazhong Agricultural University in China, and his colleagues aimed to edit genes related to symbiotic nitrogen fixation (SNF) in the legume Lotus japonicus using CRISPR/Cas9.
The team customized an sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and generated 20 T0 plants, two of them containing biallelic homozygous mutations. Due to the success of the SYMRK modification, the team then designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) to test the possibility of generating multiple gene knockouts.
Twenty out of 70 transformed plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Triple mutant knockouts of LjLbs were also obtained by stable transformation using two sgRNAs.
These results demonstrate that the CRISPR/Cas9 system can support efficient editing of single gene or simultaneous editing of multiple genes in Lotus japonicus.
For more information, read the article in Frontiers in Plant Science.
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