5-Hydroxymethylcytosine (5hmC) is a newly identified base modification in mammalian genomic DNA that can accumulate to relatively high levels in certain tissues or cells. Current sequencing methods cannot differentiate 5mC from 5hmC, however. Therefore, the immediate challenge is to develop robust methods to ascertain the positions of 5hmC within the mammalian genome. We have developed a Tet-Assisted Bisulfite Sequencing (TAB-Seq) method that allows for both genome-wide and loci-specific sequencing of 5hmC with single-base resolution and accurate abundance at modification sites. The essence of the TAB-Seq approach is to enzymatically convert 5mC to 5caC, which reads as T in bisulfite sequencing, while 5hmC can be protected through glucosylation and still reads as C. Therefore, we can differentiate 5hmC from 5mC and C in TAB-Seq. A critical step is the Tet enzyme-catalyzed conversion of 5mC to 5caC. The lower conversion rate of 5mC to 5caC, the more sequencing depths are required to differentiate 5hmC from un-converted 5mC, which could significantly reduce resolution and increase sequencing costs. A major limitation of TAB-Seq is the requirement of the highly active mTet1 that must convert at least 96% of 5mC to 5caC. The University of Chicago Office of Technology and Intellectual Property has filed a patent application for TAB-Seq and licensed the technology to Wisegene, a small, Illinois-based biotechnology company, to further develop and commercialize TAB-Seq. The kit has been commercialized, but protein aggregation problem has severely limited large-scale production of highly active Tet enzymes. In collaboration with Professor Chuan He's group at the University of Chicago, we plan to systematically engineer mouse Tet1 and Tet2 to significantly improve the stability and activity of these enzymes for large-scale production and robust application in Tab-Seq. The proposed research will develop urgently needed tools for the broad biology community to study one of the most cutting-edge frontiers of life sciences research: the potential functional roles of 5hmC in epigenetics, development, and various human diseases.