Combinatorial gene editing in mammalian cells using ssODNs and TALENs.
The regulation of gene editing is being elucidated in mammalian cells and its potential as well as its limitations are becoming evident. ssODNs carry out gene editing by annealing to their complimentary sequence at the target site and acting as primers for replication fork extension. To effect a genetic change, a large amount of ssODN molecules must be introduced into cells and as such induce a Reduced Proliferation Phenotype (RPP), a phenomenon in which corrected cells do not proliferate. To overcome this limitation, we have used TAL-Effector Nucleases (TALENs) to increase the frequency, while reducing the amount of ssODN required to direct gene correction. This strategy resolves the problem and averts the serious effects of RPP. The efficiency of gene editing can be increased significantly if cells are targeted while they progress through S phase. Our studies define new reaction parameters that will help guide experimental strategies of gene editing.
Department of Chemistry, Delaware State University, 1200 N. DuPont Highway Dover, DE 19901.
Bryan
B
Strouse
Department of Chemistry, Delaware State University, 1200 N. DuPont Highway Dover, DE 19901.
Pawel
P
Bialk
Department of Chemistry, Delaware State University, 1200 N. DuPont Highway Dover, DE 19901.
Rohina A
RA
Niamat
Department of Chemistry, Delaware State University, 1200 N. DuPont Highway Dover, DE 19901.
Natalia
N
Rivera-Torres
Department of Chemistry, Delaware State University, 1200 N. DuPont Highway Dover, DE 19901.
Eric B
EB
Kmiec
D000818:Animals; D004261:DNA Replication / Q000235:genetics*; D003851:Deoxyribonucleases / Q000235:genetics; D018390:Gene Targeting; D045325:HCT116 Cells; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry*; D015533:Transcriptional Activation
D009841:Oligonucleotides; D003851:Deoxyribonucleases