Construction and optimization of a base editor based on the MS2 system

Background:Catalytic defect Cas9‐cytosine deaminase fusion is widely used in base editing.The Multiple copy numbers of the MS2 binding site(MBS)can recruit multiple MS2 coat proteins(MCPs),which are usually applied to amplify signals.Our study aimed to apply the MS2 signal amplification system to the base editing system in order to achieve simultaneous mutations of multiple bases at the target genome site.Methods:Multiple copy numbers of the MS2 were ligated to the 3′‐end of sgRNA,and MCP was fused to the 5′‐end of cytosine deaminases.The MS2 was recognized by MCP to recruit cytosine deaminase for base substitutions(C‐T)at the target site.Different Cas9 variants,different cytosine deaminases and different copy numbers of MS2 were tested in this system,and the different versions of base editors were compared by editing efficiency and window.Results:In this study,dCas9,nCas9(D10A)and nCas9(H840A)were used.Among these 3 Cas9 variants,dCas9 exhibited higher base mutation efficiency.Two cytosine deaminases were then applied and the efficiency of rAPOBEC1 deaminase was found to be higher than AID.We also increased the copy numbers of MS2 linked to sgRNA from 2 to 12.Disappointingly,the sgRNA‐12x MS2 did not improve the editing efficiency or increase the editing window.Conclusion:An optimal version of base editor based on the MS2 system,MS2‐BErAPOBEC1(sgRNA‐2x MS2,MCP‐rAPOBEC1 and dCas9),was obtained.This tool can simultaneously mutate multiple bases at the target site,providing a new approach for the study of genome functions.

分泌包载ANXA2-MS2 RNA外泌体的稳转细胞株的构建

目的:基于MCP-MS2示踪系统构建能稳定分泌含有标签的膜联蛋白A2(ANXA2)RNA的外泌体的供体细胞株。方法:利用慢病毒载体构建稳定表达MCP-EGFP的HEK293细胞株,采用流式细胞技术筛选具有较好荧光特性的单克隆细胞株。然后再次通过慢病毒稳定表达系统构建出能分泌结合有MCP-EGFP标记的ANXA2 RNA外泌体的供体细胞株。通过透射电镜、NTA、NanoFCM、共聚焦显微镜等仪器对供体细胞所产生的外泌体进行表征。结果:通过慢病毒载体成功构建了MCP-EGFP稳定表达细胞株,然后利用该细胞株通过慢病毒载体成功导入含有MS2标签的ANXA2基因。实验结果表明,构建的供体细胞与正常细胞所分泌的外泌体形态相似,实实施时荧光定量PCR的结果显示分泌的外泌体中含有MS2特殊序列,而同时Western blot的结果显示分泌的外泌体中含有EGFP蛋白。结论:通过慢病毒法成功建立了能分泌含有MS2标记的ANXA2 RNA的外泌体供体细胞株,为高效、稳定的装载特定RNA外泌体的供体细胞株的构建提供了新的方法和理论基础。

Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/ Cas9-mediated knock-in

In mammalian cells,long noncoding RNAs(lncRNAs)form complexes with proteins to execute various biological functions such as gene transcription,RNA processing and other signaling activities.However,methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited.Here,we report the development of CERTIS(CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System)to visualize and isolate endogenous lncRNA,by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR7Cas9 technology.In this study,we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1.In addition,CERTIS displayed superior performance on both short-and long-term tracking of NEAT1 dynamics in live cells.We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors.Moreover,RNA Immunoprecipitation(RIP)of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle.Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.

植物中指导RNA偶联转录调控因子抑制基因表达研究

本研究建立了一种简单、精确和高效的新型抑制植物基因表达的技术体系。将绿色荧光蛋白(green fluorescent protein,GFP)、指导RNA(guide RNA,g RNA)和编码转录抑制因子与噬菌体外壳融合蛋白的三种基因构建到植物表达载体中。利用农杆菌介导的植物瞬时表达技术侵染本氏烟草。研究显示gfp基因在烟草叶片中的表达受到显著抑制,抑制率达到36.2%。此系统可运用于对其他基因进行基因调控,由于只需要改变g RNA,而其他组成原件保持不变,因此该技术具有操作简单及广泛的适应性等特点。为在植物基因组中精确抑制基因表达提供了有效的工具。