Study On CRISPR/Cas9 Mediated Genome Editing And Tanshinones Biosynthesis Pathway In Salvia Miltiorrhiza

The dry root and rhizome of the Lamiaceae perennial herb,Salvia miltiorrhiza Bunge,is also known as Danshen.It is widely distributed in many areas of China with a rich resource.Danshen has been used in medicine since ancient China with the effect of relieving pain,promoting the circulation of blood,and stimulate the menstrual flow,which is firstly recorded in "Shennong Ben Cao Jing".In recent years,many studies report that Danshen has a significant effect on expanding blood vessels,anti-atherosclerosis,antiphlogosis,liver protection and so on.Its pharmacological activity is largely due to the presence of the lipid-soluble compounds along with the water-soluble phenolic acids,including tanshinone I,tanshinone II A,tanshinone I IB,cryptotanshinone,tanshinol,salvianolic acid and so on.The tanshinones belong to diterpene quinone.It is synthetized through isoprenoid synthesis pathway by using the GGPP(Geranylgeranyl diphosphate)as the precursor.However,the functions of many genes downstream the GGPP involved in the pathway are still unknown,thus emphasizing the importance of an efficient way to elucidate the gene function is necessary.In recent years,CRISPR/Cas9 has been considered as an efficient tool to knock-out the genes,which paves the way for large-scale genome editing and gene function study.It can use a small RNA as the guide to recognize the targe sequence.Then the DNA will be broken by endonuclease Cas9.Compared with ZFNs and TALEN,the CRISPR/Cas9 is easier to construct that it can target various sequences by just changing the guide RNA sequence.It’s promising that the CRISPR/Cas9 mediated genome editing will accerlerate the study of functional genome study in the future.In this study,CRISPR/Cas9 is used to knock out the key genes involved in tanshinones biosynthetic pathway and to get isolated hairy root mutants.Several targets of the CRISPR/Cas9 were designed for various genes according to the genome sequence data of Salvia miltiorrhiza.The targeted genes are as follows:SmCPSl,SmCPS2,SmCPS4,SmP1D10,SmCYP76AH1,SmCYP76AH3,SmCYP76AK1,SmCYP76AK3,Sm3383,Sm4361.Silver-stained PAGE seperation was used to indentify the hairy root mutants firstly.Based on the results of direct sequencing and TA coloing sequencing,the genotype of the mutants was determined.We achieved the hairy root mutants of SmCPS1 and SmCYP76AH1 sucdessfully.The colour of the root skin of the SmCPS1 mutants showed difference compared with the wild type.To enhance the efficiency of CRISPR/Cas9 in Salvica miltiorrhiza.the experiment of condon-optimization of the CRISPR/Cas9 vectors was done.The metabolic profiles of isolated hairy root mutants of Salvia miltiorrhiza were analyzed by UPLC-ESI-qTOF-MS together with the WT(wild type)hairy roots.We found that the three predominant tanshinones(tanshinone Ⅰ,tanshinone ⅡA,and cryptotanshinone)could not be deteceted in SmCPS1 homozygous mutants according to the TIC and EIC screening.The content of the three predominant tanshinones also reduced significantly in the chimeric mutants,demonstrating that the SmCPS1 is the key gene involved in tanshinones biosynthesis pathway.In this study,a method was established as CRISPR/Cas9-mutants-secondary metabolism analysis-elucidation of gene function.We used CRISPR/Cas9 to specifically target the genes involved in secondary metabolism of tanshinones and generate hairy root mutants of Salvia miltiorrhiza.Subsequently,try to elucidate the function of the associated genes by screening the change of secondary metabolites.The establishment of this method paves the way for the study of large scale functional genes in Salvia miltiorrhiza.