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Author Notes:

Email: gang.bao@rice.edu, gbao@emory.edu

R.N.C. and G.B. conceived the research and wrote the manuscript. R.N.C designed and performed the experiments and analyzed the data. C.M.L. helped analyze the single-cell Sanger sequencing data and conceived of the indel spectrum analysis.

D.A. helped perform FACS for separating the microinjected cells. All authors have approved the manuscript.

We would like to thank Harshavardhan A. Deshmukh for his help on cell culture and nucleofection, Dr. Wilbur Lam for helpful discussions on microinjection, and Dr. Matthew Porteus for providing the L4-R4 TALEN pair and β-Ubc-GFP donor constructs.

Cell sorting was carried out at the Flow Cytometry Core Facility at Emory University Department of Pediatrics and Children’s Healthcare of Atlanta.

Subjects:

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 & Technology
  • Multidisciplinary Sciences
  • Science & 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

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

Tools:

Journal Title:

Scientific Reports

Volume:

Volume 5

Publisher:

, 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.

Copyright information:

© 2015, Macmillan Publishers Limited

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

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