High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides Hilary M. Ellis, Daiguan Yu*, Tina DiTizio, and Donald L. Court† Gene Regulation and Chromosome Biology Laboratory, Division of Basic Sciences, National Cancer Institute at Frederick, P.O. Box B, Frederick, MD 21702 Communicated by Sankar Adhya, National Institutes of Health, Bethesda, MD, April 3, 2001 (received for review February 16, 2001) Homologous DNA recombination is a fundamental, regenerative with additional markers shown: HME6, galKtyr145UAG; HME9, ⌬ ϽϾ ⌬ ϽϾ process within living organisms. However, in most organisms, galKtyr145UAG tyrTV cat; HME10, galKtyr145UAG tyrTV cat ⌬ ␭ ⌬ ϽϾ homologous recombination is a rare event, requiring a complex set (srl-recA)301::Tn10; HME25, galKtyr145UAG { gam cat}; ␭ ⌬ ϽϾ ␭ of reactions and extensive homology. We demonstrate in this HME26, galKtyr145UAG { bet cat}; HME27, galKtyr145UAG { paper that Beta protein of phage ␭ generates recombinants in ⌬exoϽϾcat}; HME31, galKϽϾcatsacB; HME36, ⌬ ϽϾ ϽϾ ϩ ϩ ϩ chromosomal DNA by using synthetic single-stranded DNAs gal galETKM; HME41, INgal (galM Ktyr145UAGT E ); ␭ ⌬ ϽϾ ⌬ϽϾ (ssDNA) as short as 30 bases long. This ssDNA recombination can HME43, galKtyr145UAG { (exo-int) cat (gam-N)}. Note be used to mutagenize or repair the chromosome with efficiencies that we are using the symbol ϽϾ to indicate a replacement that generate up to 6% recombinants among treated cells. Mech- generated by homologous recombination technology. anistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand Materials. Oligonucleotides (oligos) were provided by Life Tech- near the DNA replication fork to generate the recombinant. This nologies (Frederick, MD) as salt free but were not purified in any type of homologous recombination with ssDNA provides new special way. We note, however, that oligonucleotides obtained from avenues for studying and modifying genomes ranging from bac- some other sources were not as active as the ones we used here. The terial pathogens to eukaryotes. Beta protein and ssDNA may prove sequences of the most active of the two oligos used for curing each generally applicable for repairing DNA in many organisms. Tn10 insertion is indicated below: cysI(cw)GTGTCATTCCCGA- CTTGCCCGCTGGCGATGGCGGAAGCAGAGCGTTTCCT- omologous recombination has been described most thor- GCCGTCTTTTATCGACAACA; metC(cw)TACAACAAGCG- Houghly in the bacterium Escherichia coli. Recombination ATGTGTGAACTGGAAGGTGGCGCAGGCTGCGTGCTA- with linear DNA in E. coli is limited because the transformed TTTCCCTGCGGGGCGGCAGCGG; thrA(cc)TTGGTGA- linear DNA is rapidly degraded by the bacterial RecBCD TTTTGGCGGGGGCAGAGAGGACGGTGGCCACCTGCC- nuclease (1). Several strategies for recombination of linear CCTGCCTGGCATTGCTTTCCAGAATATCGGCAACACG; double-stranded DNA (dsDNA) fragments in RecBCD- trpC (cc) CAAAAGGCGTGATCCGTGATGATTTCGAT - defective derivatives of E. coli have been described (2, 3). CCAGCACGCATTGCCGCCATTTATAAACATTACGCTT- However, in these mutants, the frequency of recombination is CGGC; and nadA(cc)AATCTCACCCCAGCGACTAACAAA- very low and requires thousands of base pairs of homology. GTAGAAGCGGGATGCTTTGCACCGAAGCGCGCCAT- Newly developed phage-mediated recombination systems, like TTCCAGAGAAT. the bacteriophage ␭ Red system, permit efficient linear DNA recombination even in wild-type E. coli and can use short (50-bp) Construction of HME31, a Genetic Insertion. The cat-sacB cassette, homologies to generate recombinants (4–7). This ␭ Red- carrying genes for chloramphenicol resistance and sucrose mediated recombination requires the phage Gam, Exo, and Beta sensitivity, was amplified by PCR using oligos that contain functions but does not require E. coli RecA function. RecA or homologies with the galK gene. The oligos are GACCGTTA- a RecA-like function is normally central to all DNA recombi- AGCGCGATTTGTGCGCCGTCCAGCGGCAGATG͞AT- nation activities (8). The three ␭ recombination functions each CAAAGGGAAAACTGTCCA and ACTGGAAGTCGCGG- contribute to provide homologous recombination activity with TCGGAACCGTATTGCAGCAGCTTTAT͞AAAATGAGA- linear dsDNA. ␭ Gam inhibits activities of RecBCD, including CGTTGATCGGCACG. The ‘‘͞’’ indicates the junction between the nuclease activity, thus protecting electroporated linear the galK homology on the 5Ј end and the cat-sacB primers on the dsDNA from degradation (9, 10). ␭ Exo is a dsDNA-dependent 3Јend of each oligo. The PCR product was used for homologous exonuclease that digests in the 5Ј to 3Ј direction, leaving 3Ј recombination in the strain HME6. Recombinants that had overhangs that act as substrates for recombination (11). ␭ Beta inserted the cassette into galK were selected as chloramphenicol- is a single-stranded DNA (ssDNA) binding protein that pro- resistant colonies and tested as described (4). motes annealing of complementary single strands and has been shown to mediate strand exchange in vitro (12, 13). Construction of HME41, a Genetic Inversion. To create an inversion ssDNA tails are key intermediates in homologous recombi- of the gal operon, the operon was first deleted by recombination nation between dsDNAs (14, 15). We expected that in the ␭ Red with the galϽϾdel-specific oligonucleotide (see below) into system, Exo could generate 3Ј ssDNA overhangs to which Beta could bind and finish the recombination process. Here, we show Beta can promote homologous recombination between the Abbreviations: oligo(s), oligonucleotide(s); dsDNA, double-stranded DNA; ssDNA, single- stranded DNA chromosome and a synthetic ssDNA donor. *Present address: Lexicon Genetics Incorporated, 4000 Research Forest Drive, The Wood- Materials and Methods lands, TX 77381 ␭ †To whom reprint requests should be addressed at: Building 539͞Room 243, National Genotypes of Strains Used. Strains were constructed either by Cancer Institute, Frederick, MD 21702. E-mail: [email protected]. Red recombination (4), or by standard P1 transduction. HME5 ⌬ ␭ ⌬ The publication costs of this article were defrayed in part by page charge payment. This is W3110 (argF-lac)U169 { cI857 (cro-bioA)} (see Fig. 2 for article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. description of ␭). HME5 is the parent of the following strains §1734 solely to indicate this fact. 6742–6746 ͉ PNAS ͉ June 5, 2001 ͉ vol. 98 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.121164898 Downloaded by guest on September 27, 2021 strain HME5 to create HME36 ⌬galETKM. Cells carrying this gal operon deletion were selected on minimal media with 0.1% glycerol, 0.2% 2-deoxygalactose with biotin (4). Presence of the deletion was verified by PCR with flanking primers. The galϽϾdel oligonucleotide includes 41 bases of sequence up- stream of the gal operator, followed by 40 bases of sequence downstream of galM. The 81-base galϽϾdel sequence is 5ЈTC- GCGCATAAAAAACGGCTAAATTCTTGTGTAAACGA- ͞ TTCC GGCTTTATCTCATATTGTTCAAATCACCAGCA- Fig. 1. The galK DNA segment. At the top is a sequence from the galK gene AACACCGA, where the slash indicates the deletion junction. illustrating an amber mutation galKam. The sequence shown encodes amino To reinsert the gal operon in the inverted orientation, we created acids 135 through 156. The amber mutation is at tyr-145. Below is a 70-base a PCR product of the gal operon in which homology segments oligo with a sequence identical, except for one base, to the non-template found upstream (44 bases) and downstream (43 bases) flanked strand in galKam. It was used to replace the mutation with the wild-type allele. the complete operon, which was in inverted orientation with respect to these flanking chromosomal homology regions. PCR primers were 5ЈCATTCATAAACCCTCTGTTTTATAAT- demonstrating that recombination depended upon homology CACTTAATCGCGCATAAAA͞CTCATGACGAGGGCG- between the oligo and galK. TAAC and 5ЈTATGTCGGTGTTTGCTGGTGATTTGAA- To determine the minimum length of homology for recom- ͞ bination with a single-stranded oligo, we compared the ability of CAATATGAGATAAAGCC AACGGCTAAATTCTT G- ϩ TGTAAAC, where slashes indicate the novel joints between the oligos from 20 to 70 bases long to generate Gal recombinants inversion and the chromosome. This PCR product was crossed in the galKtyr145am strain HME9. Compared with the 70-base ϩ ϫ 5 8 into the Gal deletion strain HME36, generating Gal recombi- oligo, which gave 2.2 10 recombinants per 10 viable cells, the nants. The presence of the gal inversion was confirmed in four 60-, 50-, and 40-base oligos recombined less efficiently, yielding ϩ ϫ 4 8 of four Gal isolates tested by PCR with primers specific to 4–5 10 recombinants per 10 cells. Recombination efficiency dropped to 5 ϫ 103 recombinants per 108 viable cells with the either the inverted or wild-type orientation of the gal operon. ϩ Finally, the galK amber mutation replacing codon tyr-145 was 30-base oligo, and Gal colonies were near background levels introduced by recombination as described previously (4). The with a 20-base oligo. A similar requirement for homology length ␭ experiments described here used the gal operon inversion strain was observed for recombination of linear dsDNA using the ⌬ ϩ ϩ ϩ HME41, which is (argF-lac)U169 INgal(galM Ktyr145UAGT E ) Red system (4). The drop in recombination efficiency from 40 to {␭ cI857 ⌬(cro-bioA)}. 30 bases is consistent with the observation that Beta binds poorly to oligos shorter than 36 bases (17). Construction of Strains Carrying Tn10. DY374 is W3110 nadA::Tn10 Our demonstration