Targeted sequence alteration of a chromosomal locus in mouse liver

Kamiya, Hiroyuki; Uchiyama, Masayuki; Piao, Jingshu; Nakatsu, Yoshimichi; Tsuzuki, Teruhisa; Harashima, Hideyoshi

doi:10.1016/j.ijpharm.2009.12.020

Cited by 10 publications

(6 citation statements)

References 24 publications

(33 reference statements)

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“…[10][11][12][13] In this study, we used the lacZα gene from a cloning vector (M13mp18), since sequence conversion from the mutated (TAG) sequence to the "wild-type" (TCG) could be easily judged by the blue-white selection and the PstI and XbaI sites are located near the target position (Fig. 2B).…”

Section: Resultsmentioning

confidence: 99%

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Cleavage of Target DNA Promotes Sequence Conversion with a Tailed Duplex

Suzuki

Imada

Nishigaki

et al. 2016

Biological & Pharmaceutical Bulletin

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Base sequence conversion in target DNA is achieved when a 5′-tailed duplex (TD) is introduced into cells. In this study, the effects of target DNA cleavage on sequence conversion with a TD were examined. Plasmid DNAs with and without cleavage near the target position were each introduced into HeLa cells, together with the TD. The cleavage promoted the sequence alteration efficiency by ca. 7-fold. These results suggested that the sequence conversion efficiency with the TD fragment is increased when an artificial nuclease introduces cleavage near the target site.Key words sequence conversion; tailed duplex; genome editing Much attention has been paid to targeted sequence alteration, since it would provide a powerful tool for dissecting gene functions, creating animal models with mutations in genes of interest, and treating patients with diseases caused by genetic alterations. Sequence alterations have been accomplished by using nucleases, nucleic acids, and their combinations. In particular, sequence conversion using artificial nucleases is receiving considerable attention.1-5) Donor nucleic acids, cointroduced with artificial nucleases or their genes/mRNAs, have the ability to make the desired changes in nucleotide sequences.6) Meanwhile, sequence conversions without artificial nucleases have been reported. [7][8][9][10] We designed the 5′-tailed duplex (TD) DNA, consisting of a several hundred-base single-stranded (ssDNA) fragment plus an annealed oligonucleotide (Fig. 1). The long ssDNA strand is homologous to the target DNA of TD except for the sequence alteration position. The TD fragments achieved the targeted sequence conversion in cultured cells and mouse liver. 10,11) We hypothesized that the TD is a useful donor nucleic acid due to its long homologous strand and that the conversion efficiency with the TD would be increased by the cleavage of the target DNA.In this study, we examined the effects of the target DNA cleavage on the sequence conversion efficiency with the TD fragment. We found that the cleavage promoted the efficiency by ca. 7-fold in cultured cells. The results obtained in this study revealed the utility of the TD for sequence alteration (genome editing) in combination with artificial nucleases. MATERIALS AND METHODSOligodeoxyribonucleotides (ODNs) ODNs were obtained from Fasmac (Atsugi, Japan) and Eurofins Genomics (Tokyo, Japan) in purified forms. Construction of Phage and Plasmid DNAsThe pBluescript II SK(+) plasmid (Agilent Technologies, Santa Clara, CA, U.S.A.) was digested with SspI and PvuII and ligated with a linker ODN containing KpnI and SalI sites, to obtain the pBS-BamKpnSal plasmid. The 6th AAT codon in the lacZα gene in the M13mp18 DNA (TaKaRa, Otsu, Japan) was converted to the synonymous AAC codon, to eliminate the unique EcoRI site. Two novel EcoRI sites were introduced into the upstream and downstream positions of the gene, to yield the M13EcoZEco phage DNA. The 783-bp EcoRI-EcoRI fragment containing the "wild-type" lacZα gene was inserted into the pBS-BamKpnSal plasmid clea...

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Section: Resultsmentioning

confidence: 99%

“…The TD fragments achieved the targeted sequence conversion in cultured cells and mouse liver. 10,11) We hypothesized that the TD is a useful donor nucleic acid due to its long homologous strand and that the conversion efficiency with the TD would be increased by the cleavage of the target DNA.…”

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Cleavage of Target DNA Promotes Sequence Conversion with a Tailed Duplex

Suzuki

Imada

Nishigaki

et al. 2016

Biological & Pharmaceutical Bulletin

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“…[10,11,18] In this study, we compared the gene correction (sequence alteration) efficiencies when indel mismatches were present ~330 bases distant from the target position. The target base was T at position 239 (the 2nd position of codon 80, ATC) in the pSSW plasmid DNAs, and the corresponding base was G in the TD fragment (AGC) (Fig.…”

Section: Increased Correction Efficiency With Indel Mismatchesmentioning

confidence: 99%

“…[10] The naked TD fragment altered the sequence of the rpsL chromosomal transgene in mouse livers when it was delivered by hydrodynamic tail vein injection. [11][12][13] However, the gene correction efficiency was ~0.1%, and thus should be improved.…”

Section: Introductionmentioning

confidence: 99%

Insertion and Deletion Mismatches Distant from the Target Position Improve Gene Correction with a Tailed Duplex

Kamiya

Nishigaki

Ikeda

et al. 2016

Nucleosides, Nucleotides & Nucleic Acids

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A 5'-tailed duplex (TD) DNA corrects a base-substitution mutation. In this study, the effects of insertion and deletion (indel) mismatches distant from the target position on the gene correction were examined. Three target plasmid DNAs with and without indel mismatches ~330 bases distant from the correction target position were prepared, and introduced into HeLa cells together with the TD. The indel mismatches improved the gene correction efficiency and specificity without sequence conversions at the indel mismatch site. These results suggested that the gene correction efficiency and specificity are increased when an appropriate second mismatch is introduced into the TD fragment.

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“…In contrast, when the plasmid site-specifically modified with an AP site was replicated, the action-at-a-distance mutagenesis was observed in even WRN-proficient cells. He also presented his novel genome editing method without artificial endonucleases to avoid the induction of DNA strand breaks at off-target sites [5, 6].…”

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The 32nd summer school of the Research Community for Mechanisms of Mutations

Kawanishi

2019

Genes and Environ

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The 32nd summer school of the Research Community for Mechanisms of Mutations was held at Inter-University Seminar House in Hachioji city, Tokyo, from September 7 to 8, 2019. Thirty-eight people attended this annual event, and three eminent researchers were invited to discuss DNA damage induced by endogenous aldehydes, “action-at-a-distance mutagenesis” and a novel genome editing method, and DNA repair in fungi and plants. In addition to these plenary sessions, eleven participants presented their own research in oral sessions. More than half of the participants were young scientists such as graduate/undergraduate students, post-doctoral fellows and assistant professors. All members joined in enthusiastic discussions and acquired new scientific knowledge through these two days.

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Targeted sequence alteration of a chromosomal locus in mouse liver

Cited by 10 publications

References 24 publications

Cleavage of Target DNA Promotes Sequence Conversion with a Tailed Duplex

Cleavage of Target DNA Promotes Sequence Conversion with a Tailed Duplex

Insertion and Deletion Mismatches Distant from the Target Position Improve Gene Correction with a Tailed Duplex

The 32nd summer school of the Research Community for Mechanisms of Mutations

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