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

• Type of Material: Article
• Authors: Renee N. Cottle, Georgia Institute of Technology; Ciaran M. Lee, Rice University; David Archer, Emory University; Gang Bao, Emory University
• Language: English
• Date: 2015-11-12
• Publisher: Nature Publishing Group
• Publication Version: Final Published Version
• Copyright Statement: © 2015, Macmillan Publishers Limited
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1038/srep16031
• Title of Journal or Parent Work: Scientific Reports
• ISSN: 2045-2322
• Volume: 5
• Start Page: 16031
• End Page: 16031
• Grant/Funding Information: This work was supported by the National Institutes of Health as an NIH Nanomedicine Development Center Award (PN2EY018244 to GB), and the National Science Foundation Graduate Research Fellowship (DGE-0703267 to RC).
• Keywords: Science & Technology; EMBRYO MICROINJECTION; Multidisciplinary Sciences; MESSENGER-RNA; Science & Technology - Other Topics; HEMATOPOIETIC STEM/PROGENITOR CELLS; HUMAN NEURONS; GENE-TRANSFER; ZINC-FINGER NUCLEASES; MEDIATED DELIVERY; HOMOLOGOUS RECOMBINATION; DISEASE; STEM-CELLS

Abstract

Tal-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.