Efficient targeted integration directed by short homology in zebrafish and mammalian cells

Wesley A. Wierson; Jordan M. Welker; Maira P. Almeida; Carla M. Mann; Dennis A. Webster; Melanie E. Torrie; Trevor J. Weiss; Sekhar Kambakam; Macy K. Vollbrecht; Merrina Lan; Kenna C. McKeighan; Jacklyn Levey; Zhitao Ming; Alec Wehmeier; Christopher S. Mikelson; Jeffrey A. Haltom; Kristen M. Kwan; Chi Bin Chien; Darius Balciunas; Stephen C. Ekker; Karl J. Clark; Beau R. Webber; Branden S. Moriarity; Stacy L. Solin; Daniel F. Carlson; Drena L. Dobbs; Maura McGrail; Jeffrey Essner

doi:10.7554/eLife.53968

Efficient targeted integration directed by short homology in zebrafish and mammalian cells

Wesley A. Wierson, Jordan M. Welker, Maira P. Almeida, Carla M. Mann, Dennis A. Webster, Melanie E. Torrie, Trevor J. Weiss, Sekhar Kambakam, Macy K. Vollbrecht, Merrina Lan, Kenna C. McKeighan, Jacklyn Levey, Zhitao Ming, Alec Wehmeier, Christopher S. Mikelson, Jeffrey A. Haltom, Kristen M. Kwan, Chi Bin Chien, Darius Balciunas, Stephen C. EkkerKarl J. Clark, Beau R. Webber, Branden S. Moriarity, Stacy L. Solin, Daniel F. Carlson, Drena L. Dobbs, Maura McGrail, Jeffrey Essner

Biochemistry and Molecular Biology

Research output: Contribution to journal › Article › peer-review

15 Scopus citations

Abstract

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

Original language	English (US)
Article number	e53968
Pages (from-to)	1-25
Number of pages	25
Journal	eLife
Volume	9
DOIs	https://doi.org/10.7554/eLife.53968
State	Published - May 2020

ASJC Scopus subject areas

General Neuroscience
General Biochemistry, Genetics and Molecular Biology
General Immunology and Microbiology

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Wierson, W. A., Welker, J. M., Almeida, M. P., Mann, C. M., Webster, D. A., Torrie, M. E., Weiss, T. J., Kambakam, S., Vollbrecht, M. K., Lan, M., McKeighan, K. C., Levey, J., Ming, Z., Wehmeier, A., Mikelson, C. S., Haltom, J. A., Kwan, K. M., Chien, C. B., Balciunas, D., ... Essner, J. (2020). Efficient targeted integration directed by short homology in zebrafish and mammalian cells. eLife, 9, 1-25. Article e53968. https://doi.org/10.7554/eLife.53968

Wierson, WA, Welker, JM, Almeida, MP, Mann, CM, Webster, DA, Torrie, ME, Weiss, TJ, Kambakam, S, Vollbrecht, MK, Lan, M, McKeighan, KC, Levey, J, Ming, Z, Wehmeier, A, Mikelson, CS, Haltom, JA, Kwan, KM, Chien, CB, Balciunas, D, Ekker, SC , Clark, KJ, Webber, BR, Moriarity, BS, Solin, SL, Carlson, DF, Dobbs, DL, McGrail, M & Essner, J 2020, 'Efficient targeted integration directed by short homology in zebrafish and mammalian cells', eLife, vol. 9, e53968, pp. 1-25. https://doi.org/10.7554/eLife.53968

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abstract = "Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.",

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AU - Welker, Jordan M.

AU - Almeida, Maira P.

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AU - Webster, Dennis A.

AU - Torrie, Melanie E.

AU - Weiss, Trevor J.

AU - Kambakam, Sekhar

AU - Vollbrecht, Macy K.

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AU - Ming, Zhitao

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AU - Mikelson, Christopher S.

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AU - Kwan, Kristen M.

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AU - Balciunas, Darius

AU - Ekker, Stephen C.

AU - Clark, Karl J.

AU - Webber, Beau R.

AU - Moriarity, Branden S.

AU - Solin, Stacy L.

AU - Carlson, Daniel F.

AU - Dobbs, Drena L.

AU - McGrail, Maura

AU - Essner, Jeffrey

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Efficient targeted integration directed by short homology in zebrafish and mammalian cells

Abstract

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