Journal
SCIENTIFIC REPORTS
Volume 5, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/srep16031
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Funding
- National Institutes of Health as an NIH Nanomedicine Development Center Award [PN2EY018244]
- National Science Foundation Graduate Research Fellowship [DGE-0703267]
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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 beta-globin gene. We quantified nuclease induced insertions and deletions (indels) and found that, with beta-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.
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