Construction of tgw6 Mutants in Rice Based on CRISPR/Cas9 Technology

A set of tgw6 (Thousand-grain weight 6) mutants were constructed using CRISPR/Cas9 technology in this study. Three sites of 20 nt guide RNA (gRNA) targeted to the exon of TGW6 were designed and transcribed from the U3, U6a, or U6b promo- ters, respectively. The three target sites of gRNA were then ligated to the vector pYLCRISPR/Cas9-MT(I) based on golden gate cloning strategy. The recombinant plasmid was transferred to a rice cultivar, H447 (R819/Yuzhenxiang//R819 BC3F6) by Agro- bacterium-mediated transformation. Sequencing for the genomic DNA of TGW6 locus in T0 rice showed the mutagenesis fre- quency for TGW6 was more than 90%, including 51% of homozygous deletion mutations. Further analysis for the T1 mutants showed that almost all the homozygous deletion mutants improved the thousand-grain weight significantly (more than 5%). The successful tgw6 editing not only provided a series of tgw6 mutants for high and stable yield of rice but also proved that CRISPR/Cas9 is a facile and powerful means of rice genetic engineering for scientific and agricultural applications, which has