High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

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.

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.