A massively parallel in vivo assay of TdT mutants yields variants with altered nucleotide insertion biases.

Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA with random nucleotides. 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 in 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 that generated it. Using this assay, 25,623 unique TdT variants, constructed by site-saturation mutagenesis at strategic positions, were profiled. This resulted in the isolation of several altered-bias TdTs that expanded the capabilities of our TdT-based DNA recording system, Cell History Recording by Ordered Insertion (CHYRON), by increasing the information density of recording through an unbiased TdT and achieving dual-channel recording of two distinct inducers (hypoxia and Wnt) through two differently biased TdTs. Select TdT variants were also tested in vitro , revealing concordance between each variant's in vitro bias and the in vivo bias determined from the multiplexed high throughput assay. Overall, our work, and the multiplex assay it features, should support the continued development of TdT-based DNA recorders, in vitro applications of TdT, and further study of the biology of TdT.

Lineage tracing and analog recording in mammalian cells by single-site DNA writing.

Studying cellular and developmental processes in complex multicellular organisms can require the non-destructive observation of thousands to billions of cells deep within an animal. DNA recorders address the staggering difficulty of this task by converting transient cellular experiences into mutations at defined genomic sites that can be sequenced later in high throughput. However, existing recorders act primarily by erasing DNA. This is problematic because, in the limit of progressive erasure, no record remains. We present a DNA recorder called CHYRON (Cell History Recording by Ordered Insertion) that acts primarily by writing new DNA through the repeated insertion of random nucleotides at a single locus in temporal order. To achieve in vivo DNA writing, CHYRON combines Cas9, a homing guide RNA and the template-independent DNA polymerase terminal deoxynucleotidyl transferase. We successfully applied CHYRON as an evolving lineage tracer and as a recorder of user-selected cellular stimuli.