# Engineering Long ssDNA for Genome Editing Applications

> **NIH NIH R21** · UNIVERSITY OF NEBRASKA MEDICAL CENTER · 2020 · $184,813

## Abstract

Project Summary:
 Molecular reagents that can site-specifically target loci have transformed genome engineering in animals.
In particular, RNA-programmable gene targeting nucleases like Cas9 can create double stranded breaks at
defined loci, followed by homology directed repair with a donor nucleic acid molecule containing homology on
either side of the double stranded break. However, most efforts to date have focused on gene replacement for
relatively short stretches of nucleic acids. This is because site-specific insertion/replacement of multi-kilobases
long DNA fragments into mammalian genomes is technically challenging. Solving this grand challenge is
important for a number of fields that span discovery biology and biotechnology: (i.) generation of animal
models including conditional knockout mice and mice with inducible, reporters, recombinases, transcriptional
activators and transgenic models expressing long protein coding and multi-cistronic cassettes; (ii.) high
efficiency insertion of transgenes in primary cell lines at site-specific locations; (iii.) development of diverse
antibody libraries using mammalian cells; and (iv.) developing programmable, genetically engineered T cells
for immunotherapy
 To address the long-term goal of highly efficient site-specific addition of multi-kb DNA cassettes into animal
genomes, the applicants have recently published a partial solution called Easi-CRISPR (Efficient additions with
ssDNA inserts-CRISPR). The method has been used at over a dozen loci revealing robustness, high efficiency
and, moreover, versatility as it can create conditional as well as knock-in alleles. The key to the high efficiency
of DNA cassette insertion of the Easi-CRISPR method is the use of a long ssDNA donor molecule, but current
methods to prepare multi-kb linear ssDNA are insufficient for 2-10 kb inserts. Thus, the primary objective of this
project is to develop a robust and simple method for preparation of multi-kb linear ssDNA donors and to
validate long ssDNA donors with Easi-CRISPR for genome engineering in animals. The rationale for the
proposed research is guided by the urgent need for facile methods of animal genome engineering in complex
disease models and supported by the applicants’ preliminary data on ssDNA preparation and utility of Easi-
CRISPR
 The proposed research project will be carried out by pursuing two specific aims:
 1) Optimize method for preparation of long ssDNA;
 2) Test long ssDNA for generation of transgenic animals using the Easi-CRISPR approach
 This approach is innovative because it combines the key insights from different fields of biomolecular
engineering and mouse genetics in unique ways, and it is significant in providing a generalized method for
preparing long, linear ssDNA that can be broadly used by the worldwide research community for genome
engineering of large inserts at site-specific locations.

## Key facts

- **NIH application ID:** 9933032
- **Project number:** 5R21GM129559-02
- **Recipient organization:** UNIVERSITY OF NEBRASKA MEDICAL CENTER
- **Principal Investigator:** Channabasavaiah Gurumurthy
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $184,813
- **Award type:** 5
- **Project period:** 2019-07-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9933032

## Citation

> US National Institutes of Health, RePORTER application 9933032, Engineering Long ssDNA for Genome Editing Applications (5R21GM129559-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9933032. Licensed CC0.

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