Host cells deficient for mismatch repair and their use in methods for inducing homologous recombination using single-stranded nucleic acids
Inventors
Court, Donald L. • Li, Xin-Tian • Huang, Jian-Dong • Costantino, Nina • Liu, Depei
Assignees
US Department of Health and Human Services • Office of Technology Transfer
Publication Number
US-7521242-B2
Publication Date
2009-04-21
Expiration Date
2024-05-07
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Abstract
Methods are disclosed herein for inducing homologous recombination in a host cell comprising a target nucleic acid, using a single-stranded nucleic acid molecule. The single-stranded nucleic acid molecule has a sufficient number of nucleotides homologous to the target nucleic acid to enable homologous recombination with the target nucleic acid. The host cell includes a de-repressible promoter operably linked to a nucleic acid encoding a single-stranded binding protein and is deficient for mismatch repair. Isolated host cells of use in this method are also disclosed.
Core Innovation
The invention provides methods for inducing homologous recombination in host cells using single-stranded or double-stranded nucleic acids. These methods utilize host cells deficient for mismatch repair and containing a de-repressible promoter operably linked to a nucleic acid encoding a single-stranded binding protein, such as Beta, to increase recombination efficiency. The promoter can be, for example, the lambda PL promoter under the control of a temperature-sensitive repressor. Expression of Beta alone or Beta together with Exo and/or Gam recombinases enhances recombination with target nucleic acids having sufficient homology.
The problem addressed arises from limitations in in vitro restriction-ligation techniques for DNA engineering, which depend on convenient restriction sites and limit manipulable DNA fragment size to about 25 kilobases. Existing recombineering systems using lambda phage recombination proteins have improved DNA engineering, including in bacterial artificial chromosomes (BACs). However, while single-stranded oligonucleotides (SSOs) can be used to create sequence-specific mutations, the frequency of recombination using SSOs remains suboptimal, limiting efficiency. The invention aims to increase the frequency of homologous recombination in both prokaryotic and eukaryotic host cells using recombineering with SSOs via disruption or deficiency in mismatch repair pathways.
The disclosed host cells are deficient for mismatch repair through mutations in genes encoding mismatch repair proteins such as MutS, MutH, MutL, uvrD, and/or dam, thereby reducing or eliminating mismatch repair function. This deficiency prevents correction of mismatches introduced by recombination intermediates, increasing detectable recombination events. The invention includes both constitutive and transient mismatch repair deficiencies, the latter achievable by chemical treatment or antisense technologies. The recombination substrate used can be single-stranded or double-stranded nucleic acids with regions of sufficient homology, typically at least 20 to 100 nucleotides. This system increases homologous recombination efficiency significantly compared to wild-type cells.
Claims Coverage
The patent includes multiple independent claims focusing on methods to induce homologous recombination in bacterial host cells, particularly E. coli, using single- or double-stranded nucleic acids in mismatch repair-deficient hosts with induced expression of single-stranded DNA binding proteins under de-repressible promoters.
Method for inducing homologous recombination using mismatch repair-deficient bacterial cells
Introducing a single-stranded or double-stranded nucleic acid sufficiently homologous to a target into a bacterial host deficient in mismatch repair proteins MutH, MutS, uvrD, or MutL and comprising a de-repressible promoter operably linked to a nucleic acid encoding a single-stranded binding protein, followed by induction of the binding protein to induce homologous recombination.
Use of single-stranded binding proteins Beta or RecT in recombination
Employing single-stranded DNA binding proteins such as lambda Beta or RecT operably linked to a de-repressible promoter in bacterial host cells deficient for mismatch repair to effect recombination with homologous nucleic acids.
Enhanced recombination with homologous nucleic acids differing by 1 to 5 nucleotides
Method wherein single-stranded nucleic acid molecules at least 25 nucleotides long and differing from the target nucleic acid by about one to five nucleotides are used in mismatch repair-deficient bacterial host cells with induced expression of Beta or RecT to mediate homologous recombination.
Methods applicable to extrachromosomal and chromosomal targets
Inducing homologous recombination of introduced nucleic acids with target sequences present on extrachromosomal elements such as bacterial artificial chromosomes, P1 or yeast artificial chromosomes, or on the bacterial chromosome in mismatch repair-deficient hosts.
Induction of engagement of Beta protein expression via de-repressible promoters
Using de-repressible promoters, such as the lambda PL promoter, to control expression of Beta protein in mismatch repair-deficient E. coli to initiate and increase frequency of homologous recombination with introduced nucleic acids.
The independent claims collectively cover methods of inducing homologous recombination in mismatch repair-deficient bacterial hosts, particularly E. coli, by introducing homologous single- or double-stranded nucleic acids and inducing expression of ssDNA binding proteins such as Beta or RecT under the control of de-repressible promoters, enhancing recombination frequencies with targets on chromosomes or extrachromosomal DNA.
Stated Advantages
Increased frequency of homologous recombination in host cells deficient for mismatch repair, improving recombineering efficiency.
Ability to recombine with single-stranded oligonucleotides as short as about 20 to 25 nucleotides, enabling precise genetic modifications without requiring selectable markers.
Application to large DNA fragments, such as bacterial artificial chromosomes, facilitating genomic experiments previously difficult or impossible to perform.
Flexibility of use in prokaryotic and eukaryotic cells, enhancing the utility across diverse genetic engineering contexts.
Documented Applications
Modification and subcloning of large fragments of genomic DNA from bacterial artificial chromosomes (BACs).
Engineering of bacterial chromosomes and plasmids using recombineering with single-stranded or double-stranded nucleic acids.
Generation of mouse models by modification of genes in BACs for refined genomic analysis.
Induction of conditional knock-outs in eukaryotic cells by introducing site-specific recombination sites and recombinases in tissue-specific contexts.
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