一种基于调控元件的新型碱基编辑工具BE-PLUS-AID的建立及其应用

Creating a genome editing tool for regulatory elements is an important prerequisite for the functional research of non-coding sequences. CRISPR-mediated base editing technology induces base substitutions in situ, making it a useful tool for studying the non-coding regulatory elements. Regulatory element is normally composed of multiple bases, in which single base mutation is usually inadequate to affect its function. Constructed previously by the applicant, BE-PLUS is a suitable tool to study regulatory elements for its ability to destroy the regulatory element by multi-base mutagenesis with its wide window. However, because of its deaminase rAPOBEC1 is inefficient in “GC” sequences, BE-PLUS is limited in its application to study “GC” sequence enriched regulatory regions. Deaminase AID has no significant sequence preferences, but not efficient enough. Here we optimized BE-PLUS system by replacing the cytidine deaminase rAPOBEC1 with codon optimized truncate hAID to construct an advanced base editor, called BE-PLUS-AID. Next, we will verify its function by editing DNA G-quadruplex sequences in regulation regions, achieving broad window and efficient base editing with sequence compatibility in non-coding regulatory elements. If verified, BE-PLUS-AID could make a great contribution in the investigation of non-coding regulatory elements.

创建针对调控元件的基因编辑工具是研究非编码序列功能的重要前提。基于CRISPR的碱基编辑技术，因其碱基原位替换的编辑优势，成为研究非编码区调控元件的有利工具。由于调控元件由多碱基构成，针对单碱基突变通常不足以改变其功能，申请人前期构建了BE-PLUS，具备宽窗口特性，可经多碱基饱和突变破坏调控元件，更适于针对调控元件的研究。然而调控区域富含“GC”序列，BE-PLUS中的脱氨酶rAPOBEC1在“GC”序列中效率低，应用受限。已知的脱氨酶AID虽无明显序列偏好性，但编辑效率不足以满足需求。本研究拟采用密码子优化、截短的脱氨酶hAID替代BE-PLUS中的rAPOBEC1，建立新型碱基编辑工具BE-PLUS-AID，并通过编辑DNA调控元件G-四联体进行验证，完成序列兼容性、宽窗口的高效碱基编辑工具的构建，并应用于非编码区调控元件。本课题对基因组中非编码区调控元件的功能研究具有重要科学意义。

研究非编码区域内功能基因组元件的生物学功能被认为是基因组研究的下一个前沿领域之一。CRISPR介导的碱基编辑器BE-PLUS (BE-P)具有更宽的编辑窗口，是研究调控元件功能的合适工具，为以单核苷酸分辨率原位探索基因组元件提供了强大的策略。尽管如此，基于rAPOBEC1的碱基编辑器(BE)在靶向序列中对“GC”二核苷酸序列编辑效率低，BE-PLUS的应用仍然有限。为了避免这种序列偏差，我们在BE-PLUS的基础上，开发并验证了一种基于AID的新型碱基编辑器，“BE-PLUS-AID (BE-PA)”。它对靶向序列兼容性强，并在富含“GC”的位点上可进行高效和精确的碱基编辑。为了验证BE-PA的潜力，我们利用它编辑MeCP2基因启动子中功能元件的序列，以及原位调控G-四链体(G4)元件，证实了其在基因表达调控中的作用，提示在调控基因表达方面的更广泛的应用范围。我们的研究表明，BE-PA具有高效、宽窗口、无序列偏好性的特点，可以作为研究功能基因组元件的一种强有力的工具，特别是首次实现了通过碱基编辑G4达到调控基因表达的目的。