Efficient and flexible tagging of endogenous genes by homology-independent intron targeting
- Yevgeniy V. Serebrenik1,2,6,
- Stephanie E. Sansbury1,2,6,
- Saranya Santhosh Kumar1,2,
- Jorge Henao-Mejia3,4,5 and
- Ophir Shalem1,2
- 1Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA;
- 2Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA;
- 3Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA;
- 4Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA;
- 5Division of Protective Immunity, Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
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↵6 These authors contributed equally to this work.
Abstract
Genome editing tools have simplified the generation of knock-in gene fusions, yet the prevalent use of gene-specific homology-directed repair (HDR) templates still hinders scalability. Consequently, realization of large-scale gene tagging requires further development of approaches to generate knock-in protein fusions via generic donors that do not require locus-specific homology sequences. Here, we combine intron-based protein trapping with homology-independent repair-based integration of a generic donor and demonstrate precise, scalable, and efficient gene tagging. Because editing is performed in introns using a synthetic exon, this approach tolerates mutations in the unedited allele, indels at the integration site, and the addition of resistance genes that do not disrupt the target gene coding sequence, resulting in easy and flexible gene tagging.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.246413.118.
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Freely available online through the Genome Research Open Access option.
- Received January 25, 2019.
- Accepted June 12, 2019.
This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
