A Massively Parallel <i>In Vivo</i> Assay\nof TdT Mutants Yields Variants with Altered Nucleotide Insertion Biases

Terminal deoxynucleotidyl transferase (TdT) is a unique\nDNA polymerase\ncapable of template-independent extension of DNA. TdT’s <i>de novo</i> DNA synthesis ability has found utility in DNA recording,\nDNA data storage, oligonucleotide synthesis, and nucleic acid labeling,\nbut TdT’s intrinsic nucleotide biases limit its versatility\nin such applications. Here, we describe a multiplexed assay for profiling\nand engineering the bias and overall activity of TdT variants with\nhigh throughput. In our assay, a library of TdTs is encoded next to\na CRISPR-Cas9 target site in HEK293T cells. Upon transfection of Cas9\nand sgRNA, the target site is cut, allowing TdT to intercept the double-strand\nbreak and add nucleotides. Each resulting insertion is sequenced alongside\nthe identity of the TdT variant that generated it. Using this assay,\n25,623 unique TdT variants, constructed by site-saturation mutagenesis\nat strategic positions, were profiled. This resulted in the isolation\nof several altered-bias TdTs that expanded the capabilities of our\nTdT-based DNA recording system, Cell HistorY Recording by Ordered\nInsertioN (CHYRON), by increasing the information density of recording\nthrough an unbiased TdT and achieving dual-channel recording of two\ndistinct inducers (hypoxia and Wnt) through two differently biased\nTdTs. Select TdT variants were also tested <i>in vitro</i>, revealing concordance between each variant’s <i>in\nvitro</i> bias and the <i>in vivo</i> bias determined\nfrom the multiplexed high throughput assay. Overall, our work and\nthe multiplex assay it features should support the continued development\nof TdT-based DNA recorders, <i>in vitro</i> applications\nof TdT, and further study of the biology of TdT.