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

ABSTRACT: Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA. TdT's de novo DNA synthesis ability has found utility in DNA recording, DNA data storage, oligonucleotide synthesis, and nucleic acid labeling, but TdT's intrinsic nucleotide biases limit its versatility in such applications. Here, we describe a multiplexed assay for profiling and engineering the bias and overall activity of TdT variants with high throughput. In our assay, a library of TdTs is encoded next to a CRISPR-Cas9 target site in HEK293T cells. Upon transfection of Cas9 and sgRNA, the target site is cut, allowing TdT to intercept the double-strand break and add nucleotides. Each resulting insertion is sequenced alongside the identity of the TdT variant tha

SUBMITTER: Carlson CK 

PROVIDER: S-EPMC11747941 | biostudies-literature | 2024 Oct

REPOSITORIES: biostudies-literature