# Directed evolution of polymerases that can read and write extremely long sequences

> **NIH NIH R01** · UNIVERSITY OF TEXAS AT AUSTIN · 2024 · $343,817

## Abstract

Project Summary
Advances in synthetic biology have accelerated to the point where the synthesis of entire genomes is now
possible. However, the technologies for these feats are painstaking, and the production of a new chromosome
or genome requires multiple years of effort, working from small fragments to ever larger assemblies. The
cumbersome assembly process is due in large measure to the need to carry out an ordered series of hierarchical
homologous recombination steps that proceed through transformations into organisms, primarily yeast. The
speed (and ultimately scale) of large fragment assembly would be greatly improved if it were possible to routinely
amplify very long stretches of DNA (> 100 kb) in vitro. To that end, this proposal is focused on the further
development of a novel directed evolution method known as Compartmentalized Self-Replication (CSR), in
which polymerases expressed in cells in emulsions undergo thermal cycling to amplify their own genes, to
generate long read DNA polymerases that should prove capable of generating PCR amplicons > 100 kb in length,
with few errors. To achieve this goal, we propose to develop a novel library construction method that most
efficiently brings together sequence and structural domains from a variety of DNA polymerase variants to form
diverse chimeras (Aim 1.1), and to sieve these libraries using improvements to CSR that will allow us to select
for extreme processivity in yeast (Aim 1.2) and efficient error-correction (Aim 1.3). The variants that result will
be characterized for their ability to synthesize long amplicons in vitro (Aim 2.1), for their fidelity (Aim 2.2), and
for their detailed kinetic properties (Aim 2.3). Finally, to better ensure the processivity of the resultant polymerase
chimeras, we will append either DNA-binding domains (Aim 3.1) or clamps (Aim 3.2) that should lead to much
better ability to grip DNA. In addition to accelerating the ongoing revolution in genome synthesis, such long-read
polymerases should also pave the way to new sequencing technologies, including for single molecule
sequencing and for single cell sequencing.

## Key facts

- **NIH application ID:** 10746455
- **Project number:** 5R01EB027202-04
- **Recipient organization:** UNIVERSITY OF TEXAS AT AUSTIN
- **Principal Investigator:** Andrew D Ellington
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $343,817
- **Award type:** 5
- **Project period:** 2020-03-15 → 2025-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10746455

## Citation

> US National Institutes of Health, RePORTER application 10746455, Directed evolution of polymerases that can read and write extremely long sequences (5R01EB027202-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10746455. Licensed CC0.

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