The position of DNA cleavage by TALENs and cell synchronization influences the frequency of gene editing directed by single-stranded oligonucleotides.
With recent technological advances that enable DNA cleavage at specific sites in the human genome, it may now be possible to reverse inborn errors, thereby correcting a mutation, at levels that could have an impact in a clinical setting. We have been developing gene editing, using single-stranded DNA oligonucleotides (ssODNs), as a tool to direct site specific single base changes. Successful application of this technique has been demonstrated in many systems ranging from bacteria to human (ES and somatic) cells. While the frequency of gene editing can vary widely, it is often at a level that does not enable clinical application. As such, a number of stimulatory factors such as double-stranded breaks are known to elevate the frequency significantly. The majority of these results have been discovered using a validated HCT116 mammalian cell model system where credible genetic and biochemical readouts are available. Here, we couple TAL-Effector Nucleases (TALENs) that execute specific ds DNA breaks with ssODNs, designed specifically to repair a missense mutation, in an integrated single copy eGFP gene. We find that proximal cleavage, relative to the mutant base, is key for enabling high frequencies of editing. A directionality of correction is also observed with TALEN activity upstream from the target base being more effective in promoting gene editing than activity downstream. We also find that cells progressing through S phase are more amenable to combinatorial gene editing activity. Thus, we identify novel aspects of gene editing that will help in the design of more effective protocols for genome modification and gene therapy in natural genes.
Delaware State University, Department of Chemistry, Dover, Delaware, United States of America.
Natalia
N
Rivera-Torres
Delaware State University, Department of Chemistry, Dover, Delaware, United States of America.
Bryan
B
Strouse
Delaware State University, Department of Chemistry, Dover, Delaware, United States of America.
Pawel
P
Bialk
Delaware State University, Department of Chemistry, Dover, Delaware, United States of America.
Rohina A
RA
Niamat
Delaware State University, Department of Chemistry, Dover, Delaware, United States of America.
Eric B
EB
Kmiec
D018929:Cell Culture Techniques; D053837:DNA Cleavage; D004277:DNA, Single-Stranded / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D045325:HCT116 Cells; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D003851:Deoxyribonucleases