Abstract
BackgroundCRISPR-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).ResultsResults 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.ConclusionsThese 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.
Highlights
clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing and labeling has emerged as an important tool in biologic research, in regards to potential transgenic and gene therapy applications
The creativity and scale with which researchers are utilizing clustered regularly interspaced short palindromic repeat (CRISPR) sequences and Cas9 (CRISPR-associated) proteins for genomic editing has led to an explosion of possibilities in both transgenic research and gene therapy applications (Feng et al 2015; Horii et al 2013; Li et al 2015; Mou et al 2015; Nicholson et al 2015; Petersen and Niemann 2015; Seruggia and Montoliu 2014)
While CRISPRCas9 provides an elegant method to by-pass many of the concerns related to insertional mutagenesis (Zhou et al 2014), viruses are still constrained by payload capacity (Gardlik et al 2005), which could limit utility
Summary
CRISPR-Cas genome editing and labeling has emerged as an important tool in biologic research, in regards to potential transgenic and gene therapy applications. Delivery of CRISPR-Cas 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. 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. Despite the great potential CRISPR/Cas plasmids offer, there are limitations that make delivering molecular loads to target cells challenging for widespread application. Used viruses, such as adenoviruses, adeno-associated viruses, and lentiviruses, are known for having high transfection rates
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