title: Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection.
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.
authors.affiliation: Georgia Institute of Technology and Emory University, Department of Biomedical Engineering, Atlanta, GA 30332, USA.
authors.forename: Renee N
authors.initials: RN
authors.lastname: Cottle
authors.affiliation: Rice University, Department of Bioengineering, Houston, TX 77030, USA.
authors.forename: Ciaran M
authors.initials: CM
authors.lastname: Lee
authors.affiliation: Emory University School of Medicine, Department of Pediatrics, Atlanta, GA 30322, USA.
authors.forename: David
authors.initials: D
authors.lastname: Archer
authors.affiliation: Rice University, Department of Bioengineering, Houston, TX 77030, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Bao
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002460:Cell Line; D004720:Endonucleases / Q000235:genetics*; D015870:Gene Expression; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D016678:Genome; D006801:Humans; D054643:INDEL Mutation; D020014:K562 Cells; D008845:Microinjections / Q000379:methods; D016254:Mutagenesis, Insertional; D059010:Single-Cell Analysis; D055544:beta-Globins / Q000737:chemistry / Q000235:genetics*
chemical_list: D055544:beta-Globins; D004720:Endonucleases