BE3型胞嘧啶碱基编辑器在谷氨酸棒杆菌中的开发及应用

碱基编辑技术结合了CRISPR/Cas系统的靶向特异性与碱基脱氨酶的催化活性,因其不产生双链DNA断裂、不需要外源DNA模板、不依赖同源重组修复,自开发以来,便受到研究者的追捧,在哺乳动物细胞、植物、微生物等领域相继得到开发与应用。为了进一步丰富碱基编辑系统在谷氨酸棒杆菌中的应用,将鼠源胞嘧啶脱氨酶(rAPOBEC1)与nCas9蛋白融合,实现了在谷氨酸棒杆菌中C到T的编辑,编辑比例较低(0-20%);在上述融合蛋白C端添加UGI蛋白,构建BE3型胞嘧啶碱基编辑器,抑制体内的DNA碱基切除修复机制,显著的提高了碱基编辑效率,使得C到T的碱基编辑效率高达90%;为了简化操作,将双质粒碱基编辑系统优化为单质粒碱基编辑系统,并显著提高转化效率;最后通过单质粒碱基编辑系统对基因组中其他位点的编辑测试,进一步证明了BE3型碱基编辑器在谷氨酸棒杆菌中的高效性,同时发现该碱基编辑器具有较宽的编辑窗口(PAM上游-11到-19位),有助于覆盖更多的基因组靶标位点,为谷氨酸棒杆菌的基因组改造提供了更多的工具选择。

Development and Application of BE3 Cytidine Base Editor in Corynebacterium glutamicum

The base editing technology,combining the targeted specificity of CRISPR/Cas system and the catalytic activity of cytidine deaminase,has been developed and widely applied in mammalian cells,plants and microorganism,due to not introducing double-stranded DNA break,a DNA template and relying on host homologous recombination repair pathway.In order to expand the application of base editing in Corynebacterium glutamicum,C to T conversion was achieved by fusing the cytidine deaminase(rAPOBEC1)and nCas9;however,the initial editing efficiency was low(0 to 20%).The base editor BE3 was then constructed by adding the UGI protein in the C terminus of the rAPOBEC1-nCas9 fusion,which inhibited the DNA base excise repair pathway and significantly improved the editing performance with the C to T conversion efficiency up to 90%.For convenience in future applications,the dual-plasmid base editing system was simplified to singleplasmid system,and the transformation efficiency was remarkably enhanced.Finally,by using other genomic loci as editing target,the single-plasmid based BE3 base editor was proved to be powerful with high editing efficiency and broad editing window(-11 to-19 positions upstream of the PAM sequence),which was beneficial to cover more target genomic loci and provided more tools for genome engineering of C.glutamicum.