| Circular RNA(CircRNAs)are widely distributed in viruse,archaea,bacteria,fungi and mammalian cell.It functioned as a carrier of genetic material,an intermediate product of tRNA and rRNA generation,and regulated cell metabolism and protein expression.It is more stable and has a longer half-life than linear RNA,due to its 5’-end is not exposed and can’t be attacked by exonuclease in vivo,which having a great application prospect.In this study,we constructed a circularized mRNA(cmRNA)-based spider silk protein expression system;a circRNA-based multi-copy gene sequence preparation method,and a circular gRNA(cgRNA)-based CRISPR/Cas9 editing system,further broadening the application of circRNAs.Spider silk is a kind of biological material with excellent properties,wich having strength and toughness that man-made materials do not have.However,spider silk genes consist of many tandem repeats,such as Major Ampullate Spidroin 1(MaSp1)and flagelliform silk protein(FLAG),so that the construction of expression vectors is time-consuming and laborious.To solve the problem,we successfully expressed MaSp1 recombinant spider silk protein and flagelliform silklike protein(FSLP)by circular mRNA(cmRNA)method.In this study,we use the circularization mechanism of RNA cyclase to design and generate cmRNA.CmRNA encoded only one unit sequence of MaSp1 and FSLP,providing an infinite translation template for ribosome to produce long peptide containing tandem repeats.Using this technique,we successfully expressed cmRNA of MaSp1 and FSLP,and the circularization efficiency were 11.5%and 44.5%,respectively.The molecular weight of MaSp1 and FSLP recombinant spider silk proteins were more than 110 and 88 kDa,respectively.Meanwhile,Western blot and mass spectrometry also confirmed the expression of MaSp1 and FSLP.Finally,the yield liter of MaSp1 and FSLP recombinant spider silk proteins were 22.1 mg/L and 81.5 mg/L,respectively.At present,there are three main methods to construct multi-copy gene sequence expression vectors:asymmetric sticky end complementation method and isocaudamer enzyme ligation method.The copy number generated by asymmetric sticky end complementation method is random and requires multiple enzymes for enzyme digestion and ligating.The isocaudamer enzyme ligation method is also cumbersome,requiring repeated enzyme digestion and ligating.Both methods are laborious and time-consuming.To solve this problem,we designed circular RNA(circRNA)of MaSp1 and Liraglutide(Lir),and then used reverse transcriptase to generate corresponding multi-copy cDNA by traveling around circRNA.Finally,10 copies of MaSp1 and 10 copies of Lir multi-copy sequences were obtained by Taq amplification and T vector cloning,respectively.Due to the inherent properties of RNA,guide RNA(gRNA)is the most unstable component of the CRISPR/Cas9 complex and is a target for chemical modification or structural engineering to enhance system stability.While most strategies involve chemical modifications and special processes,we used an easy biotechnology to create a more stable gRNA.Since circular RNA are not easily degraded by exonuclease,we constructed circular gRNA(cgRNA)using the autocatalytic splicing mechanism of RNA cyclase ribozyme.We first optimized the formation of cgRNA in Escherichia coli(E.coli)and found that 251 cgRNA with 251 bp exogenous sequence was functional.More importantly,compared with linear gRNA,cgRNA improves the editing efficiency of base editor.We also tested the in vitro stability of cgRNAs.The stability of cgRNAs is higher than that of linear gRNAs at all tested temperatures.CgRNAs could maintain their function at 37℃ for 24 h,while linear gRNAs completely lost their function after 8 h.Later,by adding homologous arms,the circularization efficiency of 251 cgRNA reached 88.2%and the half-life reached 30 h,which was very similar to the purified 251 cgRNA.This work provides a simple and innovative strategy that greatly improves the stability of gRNA in vivo and in vitro without additional cost and effort.In conclusion,this study constructed a cmRNA-based spider silk protein expression system.The cgRNAs expression vector was constructed and cgRNAs were prepared in vitro,which improved the gene editing efficiency of gRNA in vitro and in vivo.The circRNA technique was established to prepare multi-copy gene sequences,which was beneficial to the rapid construction of multi-copy genes.In study,we exploited circRNA related technologies,further developed circRNA-based synthetic biology technologies. |
