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

> **NIH NIH R01** · UNIVERSITY OF TEXAS AT AUSTIN · 2020 · $182,969

## Abstract

Supplemental Project Summary (derived from the original, changes underlined)
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 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. The methods developed in the execution of this proposal
should also prove extremely useful for greatly improved reagents for molecular diagnostics for SARS-CoV-2. 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). Using the methods in Aim 1.2, we can produce polymerase variants that should be able to
directly participate in RT-qPCR without sample preparation, including from samples inactivated with denaturants.
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. Using the methods described in Aim 3.1, we can generate
thermostable reverse transcriptases that should prove useful for the development of isothermal amplification
assays that can be used at point-of-care, or in resource-poor settings. 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. In the current crisis,
polymerase engineering for particular functions, directed towards needs that the community has and that need
to be resolved for forward motion on testing, is a critical component of a national plan.

## Key facts

- **NIH application ID:** 10170542
- **Project number:** 3R01EB027202-01A1S1
- **Recipient organization:** UNIVERSITY OF TEXAS AT AUSTIN
- **Principal Investigator:** Andrew D Ellington
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $182,969
- **Award type:** 3
- **Project period:** 2020-09-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10170542

## Citation

> US National Institutes of Health, RePORTER application 10170542, Directed evolution of polymerases that can read and write extremely long sequences (3R01EB027202-01A1S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10170542. Licensed CC0.

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