毕赤酵母胞嘧啶碱基编辑器的设计与功能评价

Design and function evaluation of cytosine base editors in Pichia pastoris

【目的】在巴斯德毕赤酵母(Pichia pastoris)中建立一套分子靶向突变系统,为毕赤酵母的基因工程改造提供高效的编辑工具。【方法】基于规律成簇的间隔短回文重复序列/Cas9核酸酶(clustered regularly interspaced short palindromic repeats/Cas9 nuclease,CRISPR/Cas9)技术,设计并构建nCas9与胞苷脱氨酶融合表达的胞嘧啶碱基编辑器(cytosine base editor,CBE),并选择酵母基因组中富含碱基C的一段序列作为靶标以评价CBE的碱基编辑功能。电转化酵母后,利用高通量测序技术分析CBE的编辑效率及编辑模式,并进一步探究连接肽长度、融合蛋白相对位置和gRNA靶向序列(即spacer)长度等因素对CBE功能的影响。【结果】nCas9与PmCDA1融合组成的CBE能够实现毕赤酵母基因组碱基C的高效编辑。当连接肽长度为(GGGGS)10时,CBE的编辑效率最高,编辑窗口位于前间隔序列邻近基序(protospacer adjacent motif,PAM)远端的C20–C14之间,其中C18的编辑效率可达85.1%。nCas9与PmCDA1相对位置的改变对CBE的编辑效率和编辑模式的影响不大。而gRNA靶向序列长度影响着CBE的编辑效率,且gRNA靶向序列长度不能低于17 nt,但19–23 nt之间均可引导CBE对基因组的高效编辑。【结论】本研究在巴斯德毕赤酵母中构建了一套具有高效碱基编辑活性的胞嘧啶碱基编辑器,为基于毕赤酵母的基础和应用研究提供了工具支持。

[Objective]A targeted mutation system was established in Pichia pastoris to provide an efficient editing tool for genetic engineering.[Methods]A cytosine base editor(CBE)was designed with nCas9 derived from the clustered regularly interspaced short palindromic repeats/Cas9 nuclease(CRISPR/Cas9)system and a cytosine deaminase PmCDA1.A cytosine-rich sequence in the yeast genome was selected as the target to validate the CBE activity.High-throughput sequencing was employed to analyze the editing efficiency and mode of the CBE.Furthermore,we explored how factors such as the length of linker peptides,the relative position of fusion proteins,and the length of gRNA targeting sequence affect the CBE function.[Results]The CBEs established by the fusion of nCas9 and PmCDA1 were capable of editing the cytosine in the P.pastoris genome.The highest editing efficiency was achieved with a linker peptide of(GGGGS)10,and the editing window was located between C20 and C14 at the distal end of the protospacer adjacent motif.The window location at C18 achieved the maximum editing efficiency of 85.1%.Changes in the relative position of nCas9 and PmCDA1 had minor effects on the editing efficiency and modes of CBEs.However,the length of gRNA targeting sequence affected the editing efficiency of CBEs and cannot be shorter than 17 nt.The gRNA targeting sequences of 19–23 nt were capable of guiding CBEs to edit the genome.[Conclusion]This study establishes a set of CBEs with high editing efficiency in P.pastoris,which serves as a solid foundation for further genetic engineering.