High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases - PubMed (original) (raw)
High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases
Fuqiang Chen et al. Nat Methods. 2011.
Abstract
Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions.
Figures
Figure 1
ssODN design and genome editing at the human RSK2 locus. (a) The schematic shows a 125-mer ssODN (RSK2-125) donor DNA used to incorporate three mutation types into the RSK2 locus: a silent cytosine to adenine (C to A) mutation to create a silent BamHI site, a codon conversion (TGC to GTT) to create the desired cysteine to valine change in the resulting protein and a ZFN-blocking mutation (ZBM) for each ZFN arm (ZFN-L and ZFN-R). (b) Acrylamide gel separation of amplified and BamHI-digested genomic DNA from pooled K562 cells transfected with the indicated constructs or encoding the indicated proteins and collected 2 d after nucleofection. The frequency of BamHI cleavage was quantified by densitometry. Each lane represents pooled cells from an independent transfection event. M, DNA marker (Sigma). (c) Immunoblots probing the kinase activity of the RSK2 C436V mutant in K562 cells with wild-type or ssODN-mutated RSK2 C436V (three clones) treated with fmk and phorbol 12-myristate 13-acetate (PMA) to stimulate ERK that activates RSK2 as indicated. Cell extracts were immunoprecipitated with antibodies to RSK2 and the precipitates immunoblotted with antibodies to active phosphorylated hydrophobic motif of RSK2 (anti-pHM RSK2) that detect active RSK2 or to total RSK2 (anti-RSK2). Pre-precipitation cell extracts were immunoblotted with antibodies to phosphoERK (anti-pERK) that detect active ERK or to total ERK (anti-ERK).
Figure 2
Deletion of chromosomal segments using ssODNs and ZFNs at the human AAVS1 locus. (a) General ssODN design rules for deletion of chromosomal segments relative to the ZFN cut site. Sequence distal to the ZFN cleavage site (purple) and DNA sequence containing the ZFN half-site farthest from the distal deletion sequence (green) are shown. (b) ssODN sequence used to delete 5 kb upstream of the AAVS1 ZFN cut site. The ZFN binding half-site is underlined. (c) Agarose gel separation of amplified genomic DNA from K562 cells transfected with the following constructs 2 d after nucleofection (1, ssODN plus construct encoding ZFN; 2, ssODN only; and 3, construct encoding ZFN only; see supplementary note 2 for ssODN sequence). The expected fragment sizes of the wild-type and deletion alleles are indicated. PCR fragments greater than 1.5 kb were not detected under the experimental conditions. M, DNA marker (Sigma); *3′ deletion from the ZFN cut site; **5′ and 3′ deletion off the ZFN cut site by transfecting both d5-AAVS1-0.1kb and d3-AAVS1-0.1kb deletion ssODNs; supplementary note 2); all other lanes are 5′ deletions from the cut site.
Comment in
- The author file: Greg Davis.
Baker M. Baker M. Nat Methods. 2011 Sep;8(9):699. doi: 10.1038/nmeth.1677. Nat Methods. 2011. PMID: 21984998 No abstract available.
Similar articles
- Gene editing using ssODNs with engineered endonucleases.
Chen F, Pruett-Miller SM, Davis GD. Chen F, et al. Methods Mol Biol. 2015;1239:251-65. doi: 10.1007/978-1-4939-1862-1_14. Methods Mol Biol. 2015. PMID: 25408411 - Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN).
Foley JE, Yeh JR, Maeder ML, Reyon D, Sander JD, Peterson RT, Joung JK. Foley JE, et al. PLoS One. 2009;4(2):e4348. doi: 10.1371/journal.pone.0004348. Epub 2009 Feb 9. PLoS One. 2009. PMID: 19198653 Free PMC article. - Codon swapping of zinc finger nucleases confers expression in primary cells and in vivo from a single lentiviral vector.
Abarrategui-Pontes C, Créneguy A, Thinard R, Fine EJ, Thepenier V, Fournier le RL, Cradick TJ, Bao G, Tesson L, Podevin G, Anegon I, Nguyen TH. Abarrategui-Pontes C, et al. Curr Gene Ther. 2014;14(5):365-76. doi: 10.2174/156652321405140926161748. Curr Gene Ther. 2014. PMID: 25687502 - Advances in genetic modification of farm animals using zinc-finger nucleases (ZFN).
Petersen B, Niemann H. Petersen B, et al. Chromosome Res. 2015 Feb;23(1):7-15. doi: 10.1007/s10577-014-9451-7. Chromosome Res. 2015. PMID: 25596823 Review. - Genome engineering with zinc-finger nucleases.
Carroll D. Carroll D. Genetics. 2011 Aug;188(4):773-82. doi: 10.1534/genetics.111.131433. Genetics. 2011. PMID: 21828278 Free PMC article. Review.
Cited by
- Reporter Mice for Gene Editing: A Key Tool for Advancing Gene Therapy of Rare Diseases.
Li S, Brakebusch C. Li S, et al. Cells. 2024 Sep 9;13(17):1508. doi: 10.3390/cells13171508. Cells. 2024. PMID: 39273078 Free PMC article. Review. - Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor.
Jin YY, Zhang P, Liu LL, Zhao X, Hu XQ, Liu SZ, Li ZK, Liu Q, Wang JQ, Hao DL, Zhang ZQ, Chen HZ, Liu DP. Jin YY, et al. Nat Commun. 2024 Aug 10;15(1):6843. doi: 10.1038/s41467-024-50788-x. Nat Commun. 2024. PMID: 39122671 Free PMC article. - Inherited C-terminal TREX1 variants disrupt homology-directed repair to cause senescence and DNA damage phenotypes in Drosophila, mice, and humans.
Chauvin SD, Ando S, Holley JA, Sugie A, Zhao FR, Poddar S, Kato R, Miner CA, Nitta Y, Krishnamurthy SR, Saito R, Ning Y, Hatano Y, Kitahara S, Koide S, Stinson WA, Fu J, Surve N, Kumble L, Qian W, Polishchuk O, Andhey PS, Chiang C, Liu G, Colombeau L, Rodriguez R, Manel N, Kakita A, Artyomov MN, Schultz DC, Coates PT, Roberson EDO, Belkaid Y, Greenberg RA, Cherry S, Gack MU, Hardy T, Onodera O, Kato T, Miner JJ. Chauvin SD, et al. Nat Commun. 2024 Jun 1;15(1):4696. doi: 10.1038/s41467-024-49066-7. Nat Commun. 2024. PMID: 38824133 Free PMC article. - Improving the Efficiency of CRISPR Ribonucleoprotein-Mediated Precise Gene Editing by Small Molecules in Porcine Fibroblasts.
Zhao Y, Li X, Liu C, Jiang C, Guo X, Xu Q, Yin Z, Liu Z, Mu Y. Zhao Y, et al. Animals (Basel). 2024 Feb 25;14(5):719. doi: 10.3390/ani14050719. Animals (Basel). 2024. PMID: 38473105 Free PMC article. - On the genetic basis of tail-loss evolution in humans and apes.
Xia B, Zhang W, Zhao G, Zhang X, Bai J, Brosh R, Wudzinska A, Huang E, Ashe H, Ellis G, Pour M, Zhao Y, Coelho C, Zhu Y, Miller A, Dasen JS, Maurano MT, Kim SY, Boeke JD, Yanai I. Xia B, et al. Nature. 2024 Feb;626(8001):1042-1048. doi: 10.1038/s41586-024-07095-8. Epub 2024 Feb 28. Nature. 2024. PMID: 38418917 Free PMC article.
References
- Urnov FD, et al. Nature. 2005;435:646–651. - PubMed
- Bibikova M, et al. Science. 2003;300:764. - PubMed
- Porteus MH, Baltimore D. Science. 2003;300:763. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
- HHMI_/Howard Hughes Medical Institute/United States
- R01 GM071434/GM/NIGMS NIH HHS/United States
- R01 GM071434-04/GM/NIGMS NIH HHS/United States
- GM071434/GM/NIGMS NIH HHS/United States
LinkOut - more resources
Full Text Sources
Other Literature Sources