Improvement of SSO-mediated gene repair efficiency by nonspecific oligonucleotides.
Targeted gene repair mediated by single-stranded DNA oligonucleotides (SSOs) is a promising method to correct the mutant gene precisely in prokaryotic and eukaryotic systems. We used a HeLa cell line, which was stably integrated with mutant enhanced green fluorescence protein gene (mEGFP) in the genome, to test the efficiency of SSO-mediated gene repair. We found that the mEGFP gene was successfully repaired by specific SSOs, but the efficiency was only approximately 0.1%. Then we synthesized a series of nonspecific oligonucleotides, which were single-stranded DNA with different lengths and no significant similarity with the SSOs. We found the efficiency of SSO-mediated gene repair was increased by 6-fold in nonspecific oligonucleotides-treated cells. And this improvement in repair frequency correlated with the doses of the nonspecific oligonucleotides, instead of the lengths. Our evidence suggested that this increased repair efficiency was achieved by the transient alterations of the cellular proteome. We also found the obvious strand bias that antisense SSOs were much more effective than sense SSOs in the repair experiments with nonspecific oligonucleotides. These results provide a fresh clue into the mechanism of SSO-mediated targeted gene repair in mammalian cells.
Shang
Xi-Ying
XY
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College (PUMC), Department of Biochemistry, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Hao
De-Long
DL
Wu
Xue-Song
XS
Yin
Wen-Xuan
WX
Guo
Zhi-Chen
ZC
Liu
De-Pei
DP
Liang
Chih-Chuan
CC
D001483:Base Sequence; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D052416:Targeted Gene Repair / Q000379:methods*
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins