TY - JOUR
T1 - Site-specific strand bias in gene correction using single-stranded oligonucleotides
AU - Sørensen, Charlotte B
AU - Krogsdam, Anne-Margrethe
AU - Andersen, Marie S
AU - Kristiansen, Karsten
AU - Bolund, Lars
AU - Jensen, Thomas G
N1 - Keywords: Animals; Base Sequence; CHO Cells; Cricetinae; Cricetulus; DNA, Single-Stranded; Gene Targeting; Molecular Sequence Data; Oligodeoxyribonucleotides; Point Mutation; RNA; beta-Galactosidase
PY - 2004
Y1 - 2004
N2 - Targeted gene editing mediated by chimeric RNA-DNA oligonucleotides (RDOs) or single-stranded oligo-deoxyribonucleotides (ssODNs) has been demonstrated in a wide variety of cell types both in vitro and in vivo. In this study we investigated the correlation between the polarity of the used oligonucleotides and the obtained correction frequency in targeted ssODN-mediated correction of two G>A mutations (introduced at positions 659 and 1567, respectively) in an episomal beta-galactosidase gene. At position 659 the highest correction efficiency was observed using an ssODN complementary to the transcribed strand of the target gene. In contrast, at position 1567 the highest correction frequency was observed using an ssODN complementary to the nontranscribed strand of the target gene. It has been reported that site-specific gene editing mediated by ssODNs targeting the nontranscribed strand of the target gene results in a higher gene editing frequency, and it has been suggested that steric hindrance or displacement of ssODNs by traversing transcription complexes prevents efficient targeting of the transcribed strand. However, the results of the present study demonstrate that occupancy by transcriptional complexes alone does not dictate strand bias in ssODN-mediated gene editing, and that the sequences surrounding the targeted nucleotide may profoundly influence strand bias. This finding has important implications for the design of optimal ssODNs for targeted editing of a given nucleotide sequence.
AB - Targeted gene editing mediated by chimeric RNA-DNA oligonucleotides (RDOs) or single-stranded oligo-deoxyribonucleotides (ssODNs) has been demonstrated in a wide variety of cell types both in vitro and in vivo. In this study we investigated the correlation between the polarity of the used oligonucleotides and the obtained correction frequency in targeted ssODN-mediated correction of two G>A mutations (introduced at positions 659 and 1567, respectively) in an episomal beta-galactosidase gene. At position 659 the highest correction efficiency was observed using an ssODN complementary to the transcribed strand of the target gene. In contrast, at position 1567 the highest correction frequency was observed using an ssODN complementary to the nontranscribed strand of the target gene. It has been reported that site-specific gene editing mediated by ssODNs targeting the nontranscribed strand of the target gene results in a higher gene editing frequency, and it has been suggested that steric hindrance or displacement of ssODNs by traversing transcription complexes prevents efficient targeting of the transcribed strand. However, the results of the present study demonstrate that occupancy by transcriptional complexes alone does not dictate strand bias in ssODN-mediated gene editing, and that the sequences surrounding the targeted nucleotide may profoundly influence strand bias. This finding has important implications for the design of optimal ssODNs for targeted editing of a given nucleotide sequence.
U2 - 10.1007/s00109-004-0592-6
DO - 10.1007/s00109-004-0592-6
M3 - Journal article
C2 - 15517130
SN - 0946-2716
VL - 83
SP - 39
EP - 49
JO - Journal of Molecular Medicine
JF - Journal of Molecular Medicine
IS - 1
ER -