Single-strand DNA-mediated targeted mutagenesis of genomic DNA in early mouse embryos is stimulated by Rad51/54 and by Ku70/86 inhibition

Abstract

Low and variable efficiency is a major problem in targeted gene alteration, which is used as a primary tool in gene therapy and animal model studies. We tested several types of constructs alone, or in combination with other factors, to introduce a point mutation into the aB-crystallin gene in onecelled mouse embryos. We found that co-injection of ssDNA along with antibodies against Ku70/86, or supplementing the system with hRad51/hRad54, increases efficiency of targeted mutagenesis. These findings suggest that proteins in the homologous recombination DNA repair pathway contribute, and that proteins involved in the alternative nonhomologous end-joining pathway inhibit, ssDNA-mediated targeted mutagenesis. This is the first successful demonstration of targeted mutation in early mouse embryos. This novel methodology of supplying protein factors to stimulate gene modification in the nucleus has not been previously reported. Gene Therapy (2008) Keywords: targeted mutagenesis; animal models; gene correction/modification; oligonucleotide-based therapies Site-specific gene alteration by precise gene repair mechanisms is the ultimate goal in somatic gene therapy and in generating animal models of human genetic diseases via knockout/knock-in technology. Targeted genome alteration in the one-celled embryo would be ideal, however, attempts to accomplish this have not yet been successful. 1 Currently, the production of knockout and knock-in animals utilizes gene recombination techniques in mouse embryonic stem cells, followed by transferring selected stems cells into blastocysts to create a chimeric animal. Several laboratories have studied gene correction in mouse embryonic stems cells. 2,3 Even if the mutation efficiency can be increased sufficiently, the selection and screening of embryonic stem cell clones and the microinjection of selected stem cells into blastocysts are still required. Gene alteration directly in one-celled embryos will eliminate many costly and timeconsuming steps in this process. However, early mouse embryos have never been demonstrated to potentiate targeted gene alteration. This is a unique, rapidly developing and changing system distant from any other described for genomic DNA alteration. There is limited data for mouse ova and early embryos suggesting a rapid change in double-strand DNA (dsDNA) break repair pathways, 4 which could greatly affect gene correction efficiency.In other model systems, a variety of synthetic oligonucleotide constructs has been successfully used: triple helix-forming oligonucleotides (TFO), 5,6 chimeric RNA/DNA constructs, 7-10 peptide nucleic acids, 11 short DNA fragments, 12 and single-strand DNA (ssDNA). [13][14][15] However, the necessity of having a specific sequence of targeted DNA, and restrictions for triplex formation under physiological conditions, 16 impose serious limitations on the universality of TFOs as mutation mediators. Moreover, the previously reported efficiency of gene alteration mediated by chimeric RNA/DNA constructs cannot be reproduced by many e...

Gene editing activity on extrachromosomal arrays in C. elegans transgenics

“…Similar studies using mouse ova have been conducted to investigate efficiencies of in vivo single-celled embryos gene editing. Attempts to induce targeted point mutations to alter the murine genome via RNA-DNA chimeras, morpholinos (nucleic acid analogs) and single-stranded oligonucleotides to endogenous genes have been somewhat successful but still low (Morozov and Wawrousek, 2008;Tagalakis et al, 2005). Our results with extrachromosomal arrays are consistent with the previous data in germline cells and may suggest that ES cells do not contain sufficient enzymatic activity to promote gene editing unless the reaction is supplemented with pairing proteins or DNA damaging agents (Ferrara and Kmiec, 2004).…”

“…Despite many microinjection attempts on several of the transgenic strains with varying concentrations of ODNs, no gene editing events were detected. This result is consistent with previous research demonstrating the difficulties encountered in targeting embryonic stem cells and the pronuclei of single-celled embryos in mice (Dekker et al, 2003;Morozov and Wawrousek, 2008;Murphy et al, 2007;Nickerson and Colledge, 2003;Pierce et al, 2003;Tagalakis et al, 2005).…”

“…Many studies have demonstrated that mismatch repair (MMR), homologous recombination (HR), nucleotide excision repair (NER), and nonhomologous end joining (NHEJ) are involved in the regulation of gene editing. Nucleotide alteration efficiencies were detected by co-injection of proteins that either inhibited or stimulated gene editing (Morozov and Wawrousek, 2008). Co-injections of a 74-mer oligonucleotide with antibodies against Ku70/86, a DNA-binding protein involved in the nonhomologous end joining (NHEJ) pathway, resulted in a 9.9% gene editing efficiency in successfully injected embryos.…”

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

“…However, this effect could not be recapitulated in chromosomally located reporter systems, 16,23 indicating that in chromosomal targeting integration of the ssODN occurs through mechanisms that are largely independent of transcriptional activity at the target locus. Besides DNA replication and transcription, DNA repair proteins involved in homologous recombination, [34][35][36] and nucleotide excision repair, 37 have also been implicated in the targeting process. However, RNAi-mediated suppression of homologous recombination or nucleotide excision repair protein levels did not affect the targeting frequency in ESCs.…”

Over-expression of RAD51 or RAD54 but not RAD51/4 enhances extra-chromosomal homologous recombination in the human sarcoma (HT-1080) cell line

“…Thus, during naked DNA recombination (i.e., the eGFP extra-chromosomal HR assay), the effect of RAD54 is less profound than that of chromosomal DNA recombination. Morozov and Wawrousek (2007) reported that co-injection of human RAD51 and RAD54 into one-cell mouse embryos additively increases the efficiency of gene targeting. It is suggested that RAD54 promotes HR between the plasmid and chromosome.…”