# Engineering platforms for editing RNA with single base resolution

> **NIH NIH R01** · UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN · 2020 · $320,127

## Abstract

Precise modification of genomic DNA with gene editing tools is revolutionizing many scientific fields
such as biotechnology and agriculture. Despite the rapid progress in genetic engineering, two important
limitations remain that hinder widespread use of DNA editing tools in biomedicine. (1) DNA editing tools simply
introduce stochastic mutations at target sites, which often lead to gene disruption. However, correction of most
genetic diseases requires precise introduction of point mutations at target loci. (2) Gene editing tools frequently
introduce mutations at off-target sites in genomic DNA. One alternative strategy to minimize this concern is
editing RNA, which does not necessarily introduce permanent or hereditable mutations.
 Consequently, our long-term goal is to engineer tools for targeted modification of RNA with single base
resolution in human cells. Our central hypothesis is that the Pumilio and FBF (PUF) RNA binding domain can
be coupled with the cytidine deaminase APOBEC1 or the adenoside deaminase ADAR1 to introduce specific
mutations at target sites within the human transcriptome. However, the development of these tools will require
modification of some of the intrinsic biophysical properties of the three proteins.
 We will pursue our goal through three specific aims, which will test the following hypotheses: (1) Site
specific mutagenesis of key residues within the PUF architecture can be used to modulate their binding affinity
and enable the creation of PUFs that selectively bind unique sequences within the human transcriptome. (2)
PUF-APOBEC1 fusion proteins will introduce specific user-defined sequences within the human transcriptome
without off-target effects by optimizing the sequence of the linkers tethering both proteins and removing the
protein-protein interaction domains in APOBEC1. (3) Heterologous RNA complementary of a target sequence
can be used to modify specific adenines using PUFs in complex with ADAR1.
 This research is innovative because we will forward engineer proteins with distinct functions to create
novel genetic engineering tools for editing RNA, a promising new approach that has not been sufficiently
explored. These results will be significant because editing RNA with single base resolution will enable the
development of multiple gene therapies for correction of monogenic diseases or specific point mutations
causing cancer.

## Key facts

- **NIH application ID:** 9922944
- **Project number:** 5R01GM127497-03
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
- **Principal Investigator:** Pablo Perez-Pinera
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $320,127
- **Award type:** 5
- **Project period:** 2018-07-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9922944

## Citation

> US National Institutes of Health, RePORTER application 9922944, Engineering platforms for editing RNA with single base resolution (5R01GM127497-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9922944. Licensed CC0.

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