Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3

Abstract

We have previously demonstrated that site-specific insertion, deletion or substitution of one or two nucleotides in mouse embryonic stem cells (ES cells) by single-stranded deoxyribo-oligonucleotides is several hundred-fold suppressed by DNA mismatch repair (MMR) activity. Here, we have investigated whether compound mismatches and larger insertions escape detection by the MMR machinery and can be effectively introduced in MMR-proficient cells. We identified several compound mismatches that escaped detection by the MMR machinery to some extent, but could not define general rules predicting the efficacy of complex base-pair substitutions. In contrast, we found that fournucleotide insertions were largely subject to suppression by the MSH2/MSH3 branch of MMR and could be effectively introduced in Msh3-deficient cells. As these cells have no overt mutator phenotype and Msh3-deficient mice do not develop cancer, Msh3-deficient ES cells can be used for oligonucleotide-mediated gene disruption. As an example, we present disruption of the Fanconi anemia gene Fancf. Gene Therapy (2006) 13, 686-694.

Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

“…Conversely, msh6-deficient cells were permissive for +1 nt insertions, while +7 nt insertions were suppressed. The targeting frequencies of the insertion ssODNs in the Msh3-deficient ESC line 48 and the substitution ssODNs in the Msh6-deficient ESC line 8 were on average fivefold lower compared with those in the Msh2-deficient ESCs. This could be explained by the partial redundancy of MutSa and MutSb: the MSH2/MSH3 complex may suppress nucleotide substitutions to some extent in an Msh6-deficient background, whereas the MSH2/MSH6 complex may modestly suppress nucleotide insertions in an Msh3-deficient background.…”

“…Furthermore, in cell-free extracts derived from yeast and mouse embryonic fibroblasts, the efficacy of gene repair was not affected or even slightly increased upon MMR deficiency, 46,47 whereas episomal targeting frequencies in mouse embryonic fibroblasts were not significantly altered by Msh2-deficiency. 37 Although 9,48 Consistently, inactivation of the MMR pathway in E. coli through deletion of mutS, mutL or mutH enhanced the targeting frequency 2-to several 100-fold. 21,28,29,32 Although the suppressive effect of the MMR system has been most extensively studied in E. coli and mouse ESCs, MMR was also found to impose a significant barrier to ssODN-mediated gene targeting in human cell lines (Table 1).…”

“…Ablation of both heterodimeric MutS complexes by deletion of the Msh2 gene allowed effective introduction of 1-, 2-, 3-and 4-nt substitutions, as well as +1 and +4 nt insertions. 48 Deletion of the Msh3 gene only eliminates the MutSb complex, leaving the MutSa complex (MSH2/MSH6) unaffected. 49 As 4-nt insertion loops are mainly suppressed by the MSH2/MSH3 branch of MMR, these could effectively be introduced into Msh3-deficient ESCs, 48 whereas nucleotide substitutions were still suppressed by the activity of the MSH2/MSH6 complex.…”

“…48 Deletion of the Msh3 gene only eliminates the MutSb complex, leaving the MutSa complex (MSH2/MSH6) unaffected. 49 As 4-nt insertion loops are mainly suppressed by the MSH2/MSH3 branch of MMR, these could effectively be introduced into Msh3-deficient ESCs, 48 whereas nucleotide substitutions were still suppressed by the activity of the MSH2/MSH6 complex. 8 On the other hand, Msh6-deficiency only allowed the introduction of 1-, 2-, 3-and 4-nt substitutions, while nucleotide insertions were still suppressed by the activity of the remaining MSH2/MSH3 complex.…”

“…41,[51][52][53][54][55] It has been postulated that mismatches that are poorly detected by the MMR system could be introduced into MMRproficient cell lines with a reasonable efficacy. 48 Indeed, ssODNs encoding complex mismatches, such as 3-or 4-nt substitutions and 4-nt insertions, led to detectable levels of gene alteration in wild-type ESCs, but frequencies were still rather low (B10 À6 ) and general rules for ssODN design could not be established. Alternatively, temporary inhibition or downregulation of MMR proteins may limit the mutagenic effects of MMR deficiency.…”