title: CRISPR-Cas9 directed knock-out of a constitutively expressed gene using lance array nanoinjection.
abstract: BACKGROUND
  CRISPR-Cas9 genome editing and labeling has emerged as an important tool in biologic research, particularly in regards to potential transgenic and gene therapy applications. Delivery of CRISPR-Cas9 plasmids to target cells is typically done by non-viral methods (chemical, physical, and/or electrical), which are limited by low transfection efficiencies or with viral vectors, which are limited by safety and restricted volume size. In this work, a non-viral transfection technology, named lance array nanoinjection (LAN), utilizes a microfabricated silicon chip to physically and electrically deliver genetic material to large numbers of target cells. To demonstrate its utility, we used the CRISPR-Cas9 system to edit the genome of isogenic cells. Two variables related to the LAN process were tested which include the magnitude of current used during plasmid attraction to the silicon lance array (1.5, 4.5, or 6.0 mA) and the number of times cells were injected (one or three times).
  
  
  RESULTS
  Results indicate that most successful genome editing occurred after injecting three times at a current control setting of 4.5 mA, reaching a median level of 93.77 % modification. Furthermore, we found that genome editing using LAN follows a non-linear injection-dose response, meaning samples injected three times had modification rates as high as nearly 12 times analogously treated single injected samples.
  
  
  CONCLUSIONS
  These findings demonstrate the LAN's ability to deliver genetic material to cells and indicate that successful alteration of the genome is influenced by a serial injection method as well as the electrical current settings.
authors.affiliation: Department of Mechanical Engineering, Brigham Young University, Provo, UT USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Sessions
authors.affiliation: Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT USA.
authors.forename: Craig S
authors.initials: CS
authors.lastname: Skousen
authors.affiliation: Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT USA.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Price
authors.affiliation: Department of Mechanical Engineering, Brigham Young University, Provo, UT USA.
authors.forename: Brad W
authors.initials: BW
authors.lastname: Hanks
authors.affiliation: Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT USA.
authors.forename: Sandra
authors.identifier: 0000-0002-9060-0485
authors.initials: S
authors.lastname: Hope
authors.affiliation: Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT USA.
authors.forename: Jonathan K
authors.initials: JK
authors.lastname: Alder
authors.affiliation: Department of Mechanical Engineering, Brigham Young University, Provo, UT USA.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Jensen
keywords: CRISPR-Cas9; Current control; Gene knock-out; Lance array nanoinjection; Non-viral transfection; Serial injection