Engineering Tet for single-base resolution sequencing of 5hmC

描述(申请人提供)：5-羟甲基胞嘧啶(5hmC)是新发现的哺乳动物基因组DNA中的一种碱基修饰，可在某些组织或细胞中积累到相对较高的水平。然而，目前的测序方法无法区分5mC和5hmC。因此，迫切的挑战是开发可靠的方法来确定5hmC在哺乳动物基因组中的位置。我们开发了一种Tet辅助亚硫酸盐测序(TAB-Seq)方法，该方法允许对5HmC进行全基因组和位点特异性测序，具有单碱基分辨率和修饰位点的准确丰度。TAB-Seq方法的实质是将5mC转化为5caC，在亚硫酸氢盐测序中读作T，而5hmC可以通过糖基化得到保护，仍然读作C。因此，我们可以在TAB-Seq中将5hmC与5mC和C区分开来。关键的一步是Tet酶催化的5mC到5caC的转化。5mC到5caC的转化率越低，需要更多的测序深度来区分5hmC和未转化的5mC，这可能会显著降低分辨率并增加测序成本。TAB-Seq的一个主要限制是对高活性mTet1的要求，该mTet1必须至少将5mC的96%转化为5caC。芝加哥大学技术和知识产权办公室已经为TAB-Seq提交了专利申请，并将该技术授权给总部位于伊利诺伊州的小型生物技术公司Wisegene，以进一步开发TAB-Seq并将其商业化。该试剂盒已经商业化，但蛋白质聚集问题严重限制了高活性Tet酶的大规模生产。与芝加哥大学的Chuan He教授团队合作，我们计划对小鼠Tet1和TET2进行系统工程，以显著提高这些酶的稳定性和活性，以便在Tab-Seq中大规模生产和稳健应用。这项拟议的研究将为广大生物界开发急需的工具，以研究生命科学研究的最前沿领域之一：潜在的功能 5hmC在表观遗传学、发育和各种人类疾病中的作用。

DESCRIPTION (provided by applicant): 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.