| CRISPR/Cas have multiple functions,such as multi-target editing,base editing,and gene regulation,which have been used in a variety of plants.However,low efficiency of precision editing are bottlenecks of CRISPR/Cas systems,and there are few applications of CRISPR/Cas in some plants.In this study,we explored the function of Prime Editing for genome editing in the dicot Arabidopsis and achieved targeted mutagenesis in protoplasts.To improve precision editing efficiency of the Prime Editing,we tested three reverse transcriptases,which is Ca MV,Retron and MMLV-RH.The results show that all of them induced effectively targeted mutations in protoplasts and plants,and MMLV-RH improved precision editing efficiency of the Prime Editing,significantly.Meanwhile,we modified the peg RNA by adding Mpknot and Evopre Q1 sequence to its 3’ end,respectively.Mpknot also improved precision editing efficiency,significantly.Furthermore,we introduced Sp RY protein into Prime Editing and created the n Sp RY Prime Editing.The editing range was expanded to 7 types of PAM with low editing efficiency.Next,MMLV-ΔRH combined with the Mpknot version of peg RNA will be verifed.In order to develop multiple precision editing tools for multiple needs in plants,six CRISPR-Retron systems were created for targeted mutagenesis in protoplasts.No precise editing was detected.We only obtain one precise editing plant by Ec86-1 with low editing efficiency.Subsequently,a variety of modification schemes are designed to improve the efficiency of precision editing by CRISPR-Retron.By constructed CRISPR/Cas9 genome editing system in Cannabis,we induced targeted mutations of several locus of Cs THCAS1 gene in protoplasts.In order to improve the editing efficiency,a variety of optimal schemes of sg RNA were designed.Such as,9 of Pol type Ⅲ promoters were cloned from Cannabis,and Pol type Ⅱ promoters were used to improve the expression efficiency of sg RNA which contained t RNA and HH-HDV sequences.In addition,Prime Editing have been designed for precision editing in Cannabis. |
