Oligonucleotide delivery by nucleofection does not rescue the reduced proliferation phenotype of gene-edited cells.
Gene editing using single-stranded oligonucleotides (ODNs) can be used to reverse or create a single base mutation in mammalian cells. This approach could be used to treat genetic diseases caused, at least in part, by a nucleotide substitution. The technique could also be used as a tool to establish single base polymorphisms at multiple sites and thus aid in creating cell lines that can be used to define the basis for drug resistance in human cells. A troubling outcome of the gene-editing reaction is the effect on normal growth of cells that have undergone nucleotide exchange. In this work, we attempt to overcome this reduced proliferation phenotype by changing the method by which the ODN is introduced into the target cell. Using a series of assays that measure gene editing, DNA damage response, and cell viability, we report that chemically modified ODNs, the most active form of ODN for gene editing, can be used successfully if introduced into the cell by the method of nucleofection. Unlike electroporation, which has been used as the standard mode of ODN delivery, one new result is that nucleofection does not induce a dramatic loss of viability within the first 24 hours after the start of gene editing. In addition, and importantly, ODNs introduced to the cell by nucleofection do not activate the DNA damage response pathway as dramatically as ODNs introduced by electroporation. These 2 novel findings are encouraging for the application of gene editing in other systems. However, reduced proliferation phenotype is still observed when the population of corrected cells is monitored out to 8 days, and thus, delivery by nucleofection does not solve the proliferation problem encountered by cells bearing an edited gene.
Marshall Institute for Interdisciplinary Research, Marshall University, Huntington, West Virginia, USA.
Paula
P
Livingston
Bryan
B
Strouse
Haley
H
Perry
Mandula
M
Borjigin
Pawel
P
Bialk
Eric B
EB
Kmiec
D049109:Cell Proliferation; D002470:Cell Survival; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D045325:HCT116 Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics*; D010641:Phenotype; D014162:Transfection / Q000379:methods*
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins