Controlled delivery of beta-globin-targeting TALENs and CRISPR-Cas9 into mammalian cells for genome editing using microinjectionReport as inadecuate




Controlled delivery of beta-globin-targeting TALENs and CRISPR-Cas9 into mammalian cells for genome editing using microinjection - Download this document for free, or read online. Document in PDF available to download.

Journal Title:

Scientific Reports

Volume:

Volume 5

Publisher:

Nature Publishing Group | 2015-11-12, Pages 16031-16031

Type of Work:

Article | Final Publisher PDF

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.

Subjects: Health Sciences, Human Development - Engineering, Biomedical - Research Funding: 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 and Technology - Multidisciplinary Sciences - Science and Technology - Other Topics - ZINC-FINGER NUCLEASES - HEMATOPOIETIC STEM-PROGENITOR CELLS - HUMAN NEURONS - STEM-CELLS - EMBRYO MICROINJECTION - HOMOLOGOUS RECOMBINATION - MEDIATED DELIVERY - MESSENGER-RNA - GENE-TRANSFER - DISEASE -



Author: Renee N. Cottle, Ciaran M. Lee, David Archer, Gang Bao,

Source: https://open.library.emory.edu/



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