CRISPR/Cas9-mediated genome editing in Hevea brasiliensis.

Clustered regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9)has not been completely established in Hevea brasiliensis. In the present study, firstly, five endogenous U6 promoters from H. brasiliensis were identified and exploited to drive single guide RNA (sgRNA)transcription for establishing CRISPR/Cas9 transient editing system in H. brasiliensis protoplast. All five promoters were functional, however, their corresponding editing efficiencies were different and varied from 8.47 percent to 24.92 percent. Secondly, mutation profiles were characterized using 10 sgRNAs targeted five flowering time related genes (HbFT1, HbFT2 and HbTFL1?1, HbTFL1?2, HbTFL1?3)in H. brasiliensis protoplast. Three mutation patterns i. e. deletion, insertion and base substitution were detected. Among these mutations, deletion was the most prominent one and the insertions were observed only in half of 10 target sites, in which the highest insertion frequency (15 percent)occurred at TS6 target site. Base substitutions including transition and transversion were detected in 8 out of 10 target sites by deep sequencing. Lastly, stable transformation editing vector targeting phytoene desaturase gene (HbPDS)was constructed and transformed into rubber tree callus, and the "+1" bp homozygous insertions were detected for 3 out of 16 calli at the target site, which turned to expected albino phenotype in the following subculture. This study pronounced the establishment of genome editing in H. brasiliensis by CRISPR/Cas9 plasmid system.