| As a simple,inexpensive and efficient gene editing technology,CRISPR/Cas technology,is being pursuited by scientists,and has been widely used in biological related fields with remarkable achievements.Although DNA base editors have been used as "gene magic scissors" for the treatment of genetic diseases,the size limitation of their genes being embedded into r AAV vector should affect their delivery into mammalian cells.In addition,the editing efficiency of the current DNA base editors are not high enough,and the editing windows are not ideal.To address these problems,in this study we have designed and developed separate base editors based on Intein and PB1-PB2 interaction,and tested the editing efficiencies and editing windows of the modified base editors.In addition,the Escherichia coli in vivo gene editing efficiency detection methods based on ccd B and Suc B have been developed,laying a foundation for the subsequent modification of the base editors.First,an Intein-based n Cas9-CBE base editor was constructed.The n Cas9-CBE base editing system was combined with the protein trans-splicing mechanism to construct a novel Intein-based n Cas9-CBE base editor.The n Cas9 and cytosine deaminase APOBEC can be expressed independently and a fusion protein of base editor can be constructed based on Intein for base edition in vivo.Second,an E.coli CBE base editor based on the PB1-PB2 interaction was constructed.It is known that there is a highly specific interaction between the C-terminus(PB1-Cter)of PB1 from influenza virus and the N-terminus(PB2-Nter)of PB2 from influenza virus.Therefore,the PB2-Nter was fused with n Cas9 and PB1-Cter was fused with APOBEC,both of which were expressed independently,and formed protein complex through the specific interaction.In this way,the DNAs with smaller sizes could be transferred into cells and the “linker” between n Cas9 and APOBEC should be modified.Therefore,the classic base editor CBE has been modified,and the editing efficiency can be analyzed and compared with the modified base editor based on Intein.Third,a series of shuttle expression vectors were constructed to lay the foundation for convenient simultaneous base editing in E.coli and yeast.The current available base editors can only have function in one type of cells,and editing of multiple species requires the construction of different expression vectors,which brings inconvenience application.To solve this problem,in this study we have constructed a shuttle expression vector containing the base editors for expression in E.coli and Saccharomyces cerevisiae,so that protein expression and gene editing can be realized in E.coli and yeast.In the process of constructing the shuttle vector,the latest generation of Cas9 protein variant Sp RY was used.The PAM of this mutant is very flexible in addition to strong fidelity.On this basis,the shuttle plasmids were also designed and constructed to express n Cas9-Sp RY,which provided the possibility to further enhance the fidelity of gene editing under the premise of ensuring the flexibility of PAM.Fourth,the E.coli intracellular gene editing efficiency detection methods based on ccd B and Suc B genes were established.The effect of gene editing was rapidly detected by the changes in E.coli phenotype caused by the expression of ccd B and Suc B before and after gene editing.The construction of these detection systems have laid solid foundation for the subsequent directed evolution of gene editors. |
