期刊
SCIENTIFIC REPORTS
卷 5, 期 -, 页码 -出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/srep16031
关键词
-
资金
- National Institutes of Health as an NIH Nanomedicine Development Center Award [PN2EY018244]
- National Science Foundation Graduate Research Fellowship [DGE-0703267]
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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据