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LNA Modification of Single-Stranded DNA Oligonucleotides Allows Subtle Gene Modification in Mismatch-Repair-Proficient Cells

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LNA Modification of Single-Stranded DNA Oligonucleotides Allows Subtle Gene Modification in Mismatch-Repair-Proficient Cells LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells Thomas W. van Ravesteyna, Marleen Dekkera, Alexander Fishb, Titia K. Sixmab, Astrid Woltersa, Rob J. Dekkera, and Hein P. J. te Rielea,1 aDivision of Biological Stress Response, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; and bDivision of Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Edited by James E. Haber, Brandeis University, Waltham, MA, and approved January 6, 2016 (received for review July 7, 2015) Synthetic single-stranded DNA oligonucleotides (ssODNs) can be the speed of replication—had a positive effect on targeting used to generate subtle genetic modifications in eukaryotic and efficiencies, whereas replication arrest suppressed the targeting prokaryotic cells without the requirement for prior generation of efficiency (10, 11). DNA double-stranded breaks. However, DNA mismatch repair (MMR) In Escherichia coli, oligo targeting is strongly promoted by the suppresses the efficiency of gene modification by >100-fold. Here we bacteriophage λ-red–mediated recombination system (12–14). present a commercially available ssODN design that evades MMR and The phage-encoded Beta protein can bind oligonucleotides >35 enables subtle gene modification in MMR-proficient cells. The pres- nucleotides (nt) in length, thereby providing protection from ence of locked nucleic acids (LNAs) in the ssODNs at mismatching degradation and promoting annealing to complementary single- bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp stranded DNA (15–19). substitutions in MMR-proficient mouse embryonic stem cells as ef- In mammalian, yeast, and prokaryotic cell systems, it has un- fectively as in MMR-deficient cells. Additionally, in MMR-proficient equivocally been demonstrated that the DNA mismatch repair Escherichia coli, LNA modification of the ssODNs enabled effective (MMR) system has a strong suppressive effect on oligo targeting single-base-pair substitution. In vitro, LNA modification of mis- efficiencies (14, 20, 21). Disabling the MMR system through dis- GENETICS matches precluded binding of purified E. coli MMR protein MutS. ruption of the key MMR gene Msh2 increased the targeting effi- These findings make ssODN-directed gene modification particu- ciency up to 700-fold in mouse embryonic stem cells (mESCs) (20). larly well suited for applications that require the evaluation of In an E. coli mutS-deficient strain, oligo targeting was up to 500-fold a large number of sequence variants with an easy selectable more effective than in MMR-proficient bacteria (14). Apparently, phenotype. the mismatch that is created by annealing of the ssODN to its chromosomal complement creates a mismatch that is recognized by subtle gene modification | DNA mismatch repair | locked nucleic acid | the MMR system. Mismatch recognition elicits a repair reaction single-stranded oligonucleotides | embryonic stem cells that prevents incorporation of the oligonucleotide and aborts the gene modification reaction. Inconveniently, however, MMR defi- ince the rapid decline in genome sequencing costs and the ciency results in the gradual accumulation of undesired mutations – accumulation of data from genome-wide association studies, over time and may thus lead to secondary effects (22 26). Proce- S — thousands of single-nucleotide variations in disease-related genes dures that transiently suppress MMR e.g., by using RNA in- have been identified. One approach to functionally investigate terference (27, 28) or temperature-sensitive mutS and mutL — these variants of uncertain clinical significance is to introduce them mutants (29) can create a short time window of MMR deficiency into the endogenous gene of appropriate model systems that are that enables effective subtle gene modification. Nevertheless, the readily accessible to phenotypic assessment. Recently developed protocols for subtle gene modification are based on template- Significance directed repair of DNA double-strand breaks (DSBs) that are site-specifically introduced by zinc-finger, TALE, or RNA-directed With the spectacular advance of DNA-sequencing technology, a Cas9 nucleases (1). In particular, subtle mutations can effectively rapidly increasing number of subtle gene variants in the human be introduced by single-stranded DNA oligonucleotides (ssODNs) population are being identified. One approach to understanding carrying the modification of interest that serve as templates for their impact on health and disease is to re-create these variants the repair of CRISPR/Cas9-introduced DSBs (2). Several labora- in appropriate cell lines that are accessible to functional an- tories, including ours, have previously shown that ssODNs can also alyses. We present a design principle for synthetic short single- be used for subtle gene modification in the absence of DSBs. stranded DNA oligonucleotides (ssODNs) that enables effective Although less efficient than nuclease-assisted gene modification, gene modification in DNA mismatch repair (MMR)-proficient oligonucleotide-directed gene modification (also referred to as cells. We report that the placement of locked nucleic acids at “oligo targeting”)isattractiveduetoitslackofadditional mismatching bases evades MMR. Its precision, simplicity, and components, simplicity, and cost-effectiveness, especially in cost-effectiveness makes ssODN-directed gene modification cases where the consequences of the planned modifications can an attractive way of generating large numbers of sequence be scored by a selectable or easily detectable phenotype. variants, in particular when they can be evaluated by scoring Different models have been proposed for the mechanism of an easily detectable phenotype. oligo targeting (reviewed in ref. 3). Substantial evidence has been obtained that gene modification takes place during DNA repli- Author contributions: T.W.v.R., T.K.S., and H.P.J.t.R. designed research; T.W.v.R., M.D., A.F., cation (4–7). In this model, the ssODN anneals to single- A.W., and R.J.D. performed research; and T.W.v.R. and H.P.J.t.R. wrote the paper. stranded DNA in the replication fork, where it can serve as a The authors declare no conflict of interest. primer for DNA synthesis by replicative polymerases (8). This This article is a PNAS Direct Submission. process thus physically incorporates the ssODN and delivers the 1To whom correspondence should be addressed. Email: [email protected]. mutation to only one of the DNA strands (7, 9). Consistent with This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. this view, thymidine and hydroxyurea—compounds that reduce 1073/pnas.1513315113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1513315113 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 accumulation of undefined mutations cannot be totally avoided Results and may confound the phenotype of the mutation under study (28). LNA Modification Allows Single-Base-Pair Substitution in MMR- An attractive solution to this problem is to avoid the action of Proficient Cells. As readout for the efficiency of ssODN-directed MMR exclusively at the site of the targeted base substitution. gene modification, we made use of previously published MMR- − − + + Previous work in this direction has demonstrated that base an- deficient Msh2 / and MMR-proficient Msh2 / mESCs that alogs in ssODNs at the site of the substitutions can partially contain a single neomycin (neo) resistance gene, integrated into evade MMR in E. coli and in HeLa cells (30, 31). Thus far, the the Rosa26 locus, with a defective start codon, AAG (20). The use of 2′-fluorouracil and 2-aminopurine yielded a twofold in- efficiency of a 35-nt ssODN designed to generate a single A-to-T creased efficacy of substitutions to T and A, respectively, in substitution that restores the start codon and thereby confers G418 resistance was strongly suppressed by DNA MMR (Fig. 1; MMR-proficient HeLa cells (31). ref. 20). Remarkably, we found that a single LNA modification Here we present an ssODN design that allows complete eva- at the mismatching nucleotide in the ssODN resulted in an ef- − − − + + sion of MMR irrespective of the type of substitution. We dem- ficiency of ∼5 × 10 5 in both Msh2 / and Msh2 / cells (Fig. 1). onstrate that inclusion of locked nucleic acids (LNAs) in the The equal efficiencies in MMR-proficient and -deficient cells ssODNs at mismatching bases only, or also at directly adjacent indicate that a single LNA modification was sufficient to over- bases, allows effective oligo targeting in MMR-proficient cells. come the suppressive effect of MMR. An LMO with LNAs LNA nucleotides contain a modified sugar residue with an ad- flanking the mismatch evaded MMR as well and yielded an ef- − ditional 2′-C,4′-C-oxymethylene linker that confines the ribose ficiency of ∼9 × 10 5. Three consecutive LNAs (at and flanking ring to the 3′-endo conformation (32, 33). DNA:LNA duplexes the mismatching base) also evaded MMR, whereas positioning ′ ′ demonstrate an increased stability and are more resistant to LNAs at the 5 and 3 termini of the ssODN had no effect. We nucleases (34, 35). We reportthatLNA-modifiedssODNs conclude that LNA modification at or in the direct vicinity of the (LMOs) yielded equal efficiencies in MMR-proficient and mismatching nucleotide was required for MMR evasion. -deficient mESCs for the substitution of one to three bases.
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