Precise modification of genomic DNA with gene editing tools is revolutionizing many scientific fields such as biotechnology and agriculture. Despite the rapid progress in genetic engineering, two important limitations remain that hinder widespread use of DNA editing tools in biomedicine. (1) DNA editing tools simply introduce stochastic mutations at target sites, which often lead to gene disruption. However, correction of most genetic diseases requires precise introduction of point mutations at target loci. (2) Gene editing tools frequently introduce mutations at off-target sites in genomic DNA. One alternative strategy to minimize this concern is editing RNA, which does not necessarily introduce permanent or hereditable mutations. Consequently, our long-term goal is to engineer tools for targeted modification of RNA with single base resolution in human cells. Our central hypothesis is that the Pumilio and FBF (PUF) RNA binding domain can be coupled with the cytidine deaminase APOBEC1 or the adenoside deaminase ADAR1 to introduce specific mutations at target sites within the human transcriptome. However, the development of these tools will require modification of some of the intrinsic biophysical properties of the three proteins. We will pursue our goal through three specific aims, which will test the following hypotheses: (1) Site specific mutagenesis of key residues within the PUF architecture can be used to modulate their binding affinity and enable the creation of PUFs that selectively bind unique sequences within the human transcriptome. (2) PUF-APOBEC1 fusion proteins will introduce specific user-defined sequences within the human transcriptome without off-target effects by optimizing the sequence of the linkers tethering both proteins and removing the protein-protein interaction domains in APOBEC1. (3) Heterologous RNA complementary of a target sequence can be used to modify specific adenines using PUFs in complex with ADAR1. This research is innovative because we will forward engineer proteins with distinct functions to create novel genetic engineering tools for editing RNA, a promising new approach that has not been sufficiently explored. These results will be significant because editing RNA with single base resolution will enable the development of multiple gene therapies for correction of monogenic diseases or specific point mutations causing cancer.