Development of A Directed Evolution Platform for RNA Methyltransferases. - PROJECT SUMMARY N6-methyladenosine (m6A) is one of the most abundant chemical modifications in human messenger RNA (mRNA). m6A in mRNA is dynamically regulated by the effector proteins that install, remove, and specifically recognize m6A-modified transcripts, which are known as writers, erasers, and readers, respectively. Growing evidence has suggested the significant roles of m6A in cancers including acute myeloid leukemia, endometrial cancer, breast cancer, and glioblastoma. Dysregulations in m6A, its effector proteins, and related metabolites have been shown to significantly influence the development of tumors, drug resistance, prognosis, and the spread of cancer in various forms. However, understanding of the explicit roles of m6A in cancer biology remains incomplete. Current efforts on understanding epitranscriptomic regulations heavily rely on altering the expression of an effector protein, which typically generates global alterations to the modification levels on hundreds of mRNA substrates and perturbs all other molecular interactions involving the effector protein beyond their roles in regulating RNA modifications. With such convoluted results, it is challenging to deduce the explicit functions of m6A on specific genes. To identify key regulatory m6A-modified transcripts in cancers, tools that enable efficient and precise installation of m6A(s) at any one or multiple mRNAs of interest are highly desirable. One major challenge is that the existing m6A writer or methyltransferases suffer from slow turnover rates, dependence upon auxiliary proteins, and sequence biases of RNA substrates. Here we propose to develop a directed evolution platform to evolve RNA MTases with improved catalytic efficiency and reduced substrate sequence recognition biases. This directed evolution platform will leverage in vitro compartmentalization and m6A-specific endoribonucleases to achieve rapid enrichments of functional MTase variants from a diverse (1010) library. The evolved MTases will be further fused with RNA-targeting CRISPR-Cas systems to achieve site-specific installation of m6A with high efficiency and reduced off-target editing.
