Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection

dc.citation.journalTitleScientific Reports
dc.citation.volumeNumber5
dc.contributor.authorCottle, Renee N.
dc.contributor.authorLee, Ciaran M.
dc.contributor.authorArcher, David
dc.contributor.authorBao, Gang
dc.date.accessioned2016-04-04T18:26:23Z
dc.date.available2016-04-04T18:26:23Z
dc.date.issued2015
dc.description.abstractTal-effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are genome editing tools with unprecedented potential. However, the ability to deliver optimal amounts of these nucleases into mammalian cells with minimal toxicity poses a major challenge. Common delivery approaches are transfection- and viral-based methods; each associated with significant drawbacks. An alternative method for directly delivering genome-editing reagents into single living cells with high efficiency and controlled volume is microinjection. Here, we characterize a glass microcapillary-based injection system and demonstrate controlled co-injection of TALENs or CRISPR/Cas9 together with donor template into single K562 cells for targeting the human β-globin gene. We quantified nuclease induced insertions and deletions (indels) and found that, with β-globin-targeting TALENs, similar levels of on- and off-target activity in cells could be achieved by microinjection compared with nucleofection. Furthermore, we observed 11% and 2% homology directed repair in single K562 cells co-injected with a donor template along with CRISPR/Cas9 and TALENs respectively. These results demonstrate that a high level of targeted gene modification can be achieved in human cells using glass-needle microinjection of genome editing reagents.
dc.identifier.citationCottle, Renee N., Lee, Ciaran M., Archer, David, et al.. "Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection." <i>Scientific Reports,</i> 5, (2015) Macmillan Publishers Limited: http://dx.doi.org/10.1038/srep16031.
dc.identifier.doihttp://dx.doi.org/10.1038/srep16031
dc.identifier.urihttps://hdl.handle.net/1911/88832
dc.language.isoeng
dc.publisherMacmillan Publishers Limited
dc.rightsThis work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material.
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.titleControlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection
dc.typeJournal article
dc.type.dcmiText
dc.type.publicationpublisher version
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