Developing a T>A Base Editor via Protein Universe Mining and optimizing translesion polymerase

A engineering Pol η variants offers the exciting prospect of further tailoring its activity for base editing. Improving its processivity could ensure that Pol η completes the bypass of the AP site and perhaps extends a few more bases before dissociating, thus preventing the recruitment of other, potentially less desirable, cellular polymerases or repair factors. Base on this hypothesis, We decide to develop a novel T-to-A base editor. Seventy-two hours after transfection, HeLa cells were washed with PBS and digested with TRIzol (Takara). Around 5x106 cells for mRNA prepared. A total of 1.5 mg of RNA per sample was adopted as input material to get preparations for the sample. Sequencing libraries were generated by AZENTA SZ NGS LABORATORY with the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) following the manufacturerʼs instructions. The sequence library was purified (Agencourt AMPure XP Beads), and the quality was evaluated on a Bioanalyzer (Agilent High Sensitivity Chip). According to the manufacturerʼs instructions, index-coded samples were clustered by a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina), then the library was sequenced on an Illumina NovaSeq 6000 platform, and 125–150-bp paired-end reads were obtained.