Development and testing of a versatile genome editing application reporter (V-GEAR) system

Evan W. Kleinboehl; Kanut Laoharawee; Walker S. Lahr; Jacob D. Jensen; Joseph J. Peterson; Jason B. Bell; Beau R. Webber; Branden S. Moriarity

Molecular Therapy: Methods & Clinical Development (Jun 2024)

Development and testing of a versatile genome editing application reporter (V-GEAR) system

Evan W. Kleinboehl,
Kanut Laoharawee,
Walker S. Lahr,
Jacob D. Jensen,
Joseph J. Peterson,
Jason B. Bell,
Beau R. Webber,
Branden S. Moriarity

Affiliations

Evan W. Kleinboehl: Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
Kanut Laoharawee: Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
Walker S. Lahr: Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
Jacob D. Jensen: Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
Joseph J. Peterson: Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
Jason B. Bell: Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
Beau R. Webber: Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA; Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
Branden S. Moriarity: Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA; Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA; Corresponding author: Branden S. Moriarity, Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA.

Journal volume & issue: Vol. 32, no. 2
p. 101253

Abstract

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CRISPR-Cas9 and novel cas fusion proteins leveraging specific DNA targeting ability combined with deaminases or reverse transcriptases have revolutionized genome editing. However, their efficacy heavily relies upon protein variants, targeting single guide RNAs, and surrounding DNA sequence context within the targeted loci. This necessitates the need for efficient and rapid screening methods to evaluate these editing reagents and designs. Existing plasmid-based reporters lack flexibility, being fixed to specific DNA sequences, hindering direct comparisons between various editing approaches. To address this, we developed the versatile genome editing application reporter (V-GEAR) system. V-GEAR comprises genes detectable after desired editing via base editing, prime editing, or homology-directed repair within relevant genomic contexts. It employs a detectable synthetic cell surface protein (RQR8) followed by a customizable target sequence resembling genomic regions of interest. These genes allow for reliable identification of corrective editing and cell enrichment. We validated the V-GEAR system with base editors, prime editors, and Cas9-mediated homology-directed repair. Furthermore, the V-GEAR system offers versatility by allowing transient screening or stable integration at the AAVS1 safe harbor loci, rapidly achieved through immunomagnetic isolation. This innovative system enables direct comparisons among editing technologies, accelerating the development and testing of genome editing approaches.

Published in Molecular Therapy: Methods & Clinical Development

ISSN: 2329-0501 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Biology (General): Genetics; Science: Biology (General): Cytology
Website: http://www.cell.com/molecular-therapy-family/methods/latest-content

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