A Rapid and General Assay for Monitoring Endogenous Gene ...

Chapter 15 A Rapid and General Assay for Monitoring Endogenous Gene Modification Dmitry Y. Guschin, Adam J. Waite, George E. Katibah, Jeffrey C. Miller, Michael C. Holmes, and Edward J. Rebar Abstract The development of zinc finger nucleases for targeted gene modification can benefit from rapid functional assays that directly quantify activity at the endogenous target. Here we describe a simple procedure for quantifying mutations that result from DNA double-strand break repair via non-homologous end joining. The assay is based on the ability of the Surveyor nuclease to selectively cleave distorted duplex DNA formed via cross-annealing of mutated and wild-type sequence. Key words: Zinc finger nuclease (ZFN), designed zinc finger proteins, non-homologous end joining (NHEJ), Surveyor nuclease, Cel 1, genome modification.

1. Introduction Designed zinc finger nucleases (ZFNs) promise to broadly enable genome engineering of higher eukaryotes. Recent studies have demonstrated the ability of these proteins to catalyze efficient modification of endogenous genes (up to 50% (1)) in diverse species and cell types (1–13). Designed ZFNs may be developed and assayed using a variety of methods (for example see (4, 7, 14–17)), but to be useful for genome engineering they must efficiently cleave the target locus in the desired cells. Since context-specific factors such as transcriptional status and chromatin structure can limit the ability of reporters to predict endogenous function, an assay that monitors activity directly at

J.P. Mackay, D.J. Segal (eds.), Engineered Zinc Finger Proteins, Methods in Molecular Biology 649, DOI 10.1007/978-1-60761-753-2_15, © Springer Science+Business Media, LLC 2010

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the endogenous target provides the most reliable means for identifying and comparing ZFN candidates. Genome engineering strategies generally take advantage of one of the two major DNA repair pathways – homology-directed repair (HDR) or non-homologous end joining (NHEJ) – to modify target sequences (18, 19). The choice of strategy constrains options for quantifying modification. HDR-based approaches, for example, copy sequence from a “donor” DNA into the genome and, therefore, may be designed to introduce features that facilitate quantitation such as a new restriction site or size-resolvable insert (5–7). However, screening for HDRmediated modifications can be cumbersome given the need to construct and validate a new donor with homology arms specific for each ZFN target site. Moreover, assays that PCR amplify the targeted locus must be carefully designed to avoid artifacts caused by the presence of the (typically) large amount of residual donor DNA. Donor-free strategies for genome engineering utilize the other major DNA repair pathway, NHEJ, to bring about targeted mutation. These approaches yield a heterogeneous mix of minor insertions and deletions that are consequently more difficult to monitor than the discrete modifications introduced via HDR (for example see (20)). Such events may be quantified by sequencing the targeted locus, although cost and throughput considerations can render this approach impractical for quantifying low modification levels and/or a large number of samples. Alternatively, one may monitor disruption of an overlapping restriction site (4, 8, 13). This strategy relies on the ability of the minor mutations introduced by NHEJ to confer resistance to digestion by a restriction enzyme whose target overlaps the site of the ZFN-induced break. Although rapid and simple, this approach lacks generality, as it requires fortuitous positioning of a restriction site at the intended target. In this chapter, we describe an alternative approach for monitoring the heterogeneous mix of mutations introduced via NHEJ. Our assay uses the Surveyor nuclease (Transgenomic) to selectively digest mismatched duplex, which allows one to detect and estimate the mutated fraction of an amplicon pool after crossannealing with wild-type sequences (21). Figure 15.1 provides an overview of the assay procedure in the context of a typical gene modification study. After cellular expression of ZFNs (see Notes 1 and 2) and subsequent mutagenesis, the targeted locus is PCR amplified (Section 3.1). The amplicons are then reannealed, which converts any mutations into mismatched duplexes, and digested with the Surveyor nuclease, which selectively cleaves distorted duplex DNA (Section 3.2) (22). Finally, digestion products are resolved via PAGE and quantified using a gel imaging station. Data analysis is performed on the digital gel image

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ZFN induced mutagenesis ZFNs digest genomic DNA

NHEJ generates mutations

Suveyor assay PCR amplify targeted locus

mismatched heteroduplex

Denature and reanneal

cleaved heteroduplex

Digest with Suveyor nuclease

Treated Resolve on PAGE

Control uncleaved cleaved

Fig. 15.1. Overview of procedures. Top panel: A typical gene modification experiment initiates with ZFN expression in an appropriate cell type. ZFNs cleave their endogenous target, and subsequent repair yields minor mutations in a subset of chromosomes (in this case 20%). A ZFN dimer is pictured schematically at the top of the panel bound to genomic DNA (straight double lines) and the resultant cleavage event is depicted by a gap. NHEJ-induced mutations in the genomic DNA are denoted by jagged double lines. Bottom panel: The Surveyor assay initiates with cell lysis and PCR amplification of the targeted locus. Cross-annealing of mutated and wild-type sequence then converts mutations into mismatched duplexes. The reannealed amplicon is digested with the Surveyor nuclease, which selectively cleaves distorted duplex DNA (22). Digestion products are then resolved via PAGE. Horizontal half arrows indicate PCR primers; straight double lines and jagged lines denote, respectively, wild-type and mutated amplicon sequences. The rectangle at bottom schematically depicts a polyacrylamide gel with banding patterns yielded by ZFN-treated and control cells.

(Section 3.3). The assay is sensitive down to ~1% gene modification and yields values that correlate with mutation rates obtained from direct cloning and sequencing of the target locus. The assay is rapid, inexpensive, and as convenient as a restriction digest.

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2. Materials 2.1. Preparation and PCR Amplification of Genomic DNA

1. QuickExtract DNA extraction solution (Epicentre Biotechnologies, QE09050). 2. AccuPrime taq DNA polymerase (Invitrogen, 12346-086). 3. 96-well PCR reaction plates. 4. PCR thermocycler. 5. PCR primers (for design considerations, see Note 3). 6. 1% precast agarose gel (Biorad, 161-3063).

2.2. Surveyor Digest and Polyacrylamide Gel Electrophoresis (PAGE)

1. Surveyor nuclease S (Transgenomic, 706025). 2. 10% acrylamide Criterion TBE gel (26-well, BioRad, 345-0053). 3. 10 mg/mL ethidium bromide solution (Sigma-Aldrich, E1510). 4. UV imaging station (e.g., Fluorochem SP, Alpha Innotech). 5. Gel analysis software (e.g., ImageQuant 5.1, GE Healthcare). 6. 5× gel loading buffer: 15% Ficoll-400, 0.05% Orange G (Sigma-Aldrich, 46327 and O3756). 7. Multi flex round gel tips (Sorenson, 13790). 8. 5–50 μL eight-channel pipettor (ThermoFisher, 4610120). 9. 1× TBE gel running buffer: prepare by diluting 10× TBE stock (Fisher BioReagents, BP13334)

3. Methods 3.1. Preparation and PCR Amplification of Genomic DNA

1. Pellet 106 ZFN-treated cells at 270×g for 5 min. 2. Gently remove the supernatant. 3. Add 100 μL of QuickExtract solution and pipette vigorously (see Note 4). 4. Transfer the reaction to PCR-compatible tubes and finish the genomic DNA extraction by incubating at 68◦ C for 15 min, followed by 95◦ C for 8 min. Then hold at 4◦ C. 5. Set up a 50 μL PCR reaction using the AccuPrime Taq DNA Polymerase High Fidelity kit: 5 μL of 10× Accuprime buffer II, 2 μL of template DNA from step 4, 2.5 μL of each primer at 10 μM, 0.2 μL of AccuPrime Taq DNA Polymerase High Fidelity, and sterile water to 50 μL (see Note 5).


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6. Run the PCR as follows: 95◦ C for 5 min; 35× (95◦ C for 30 s, 60◦ C for 30 s, 68◦ C for 40 s); 68◦ C for 2 min; hold at 4◦ C. 7. Verify the amplification by running 5 μL of PCR reaction on an agarose gel. 3.2. Surveyor Digest and Gel Electrophoresis

1. Mix 6 μL of 1× Accuprime buffer II with 3 μL of PCR product and incubate as follows: 95◦ C for 5 min, 95–85◦ C at –2◦ C/s, 85–25ºC at –0.1◦ C/s; hold at 4◦ C. This step melts and randomly reanneals the amplicons, which converts any mutations into mismatched duplex DNA. 2. Store the annealed samples on ice. Add 1 μL of Surveyor nuclease S per sample. 3. Incubate at 42◦ C for 20 min in a PCR machine. 4. Add to each reaction 3 μL of 5× gel loading buffer. 5. Load the samples on a 10% acrylamide BioRad Criterion TBE gel. 6. Run the gel at 100 V until the Orange G dye front reaches bottom. 7. Stain the gel with 5 μL of ethidium bromide solution in 100 mL of 1× TBE gel running buffer for 10 min, then wash the gel quickly three times with 100 mL of deionized water (100 bp. 4. Although the protocols described here utilize tissue culture samples processed using QuickExtract solution (Epicentre Biotechnologies) as the source of genomic DNA, we note that the Surveyor assay can be used to quantify mutation levels in DNA from any source. For example, in other studies we have successfully used genomic DNA from transgenic fish (3) and isolated using alternative purification kits (21) as template for the assay. 5. We have observed that the lysate can inhibit PCR, so we typically use no more than 2 μL of lysate per 50 μL of reaction. 6. We occasionally observe cleavage products that are not ZFN dependent (see Fig. 15.2 lane “9–”), which we attribute to the presence of polymorphisms or intrinsically distorted sequence. In many such cases, one may still estimate mutation rates if the ZFN-dependent bands migrate away from the ZFN-independent band. PCR primers may also be redesigned to reposition the amplicon to exclude the Surveyor-susceptible sequence. 7. Accurate quantitation rests on four assumptions: (i) complete duplex melting, (ii) random strand reassortment during the reannealing step, (iii) a mutation spectrum of sufficient diversity to prevent significant annealing of identically mutated DNA strands, and (iv) complete cleavage of all mismatches by the Surveyor enzyme. To the extent that conditions deviate from (i) to (iv), the assay will tend to underestimate mutation levels. In practice, we have found that the methods described in this chapter typically agree with mutation levels gauged by sequencing, with underestimates by up to a factor of 2 for a minority of ZFNs. 8. In some instances, a ZFN-treated lane will contain visibly obvious undigested heteroduplex running above the parent amplicon (e.g., the faint band above the parent band in Fig. 15.2 lanes 3+, 7+, and 8+). If present, this band is quantified separately and the fraction cleaved is calculated as in step 2 of Section 3.3 using the formula fraction cleaved = density from (ii) + heteroduplex density density from (i) + heteroduplex density Such samples may also be re-assayed using a higher level of Surveyor enzyme or lower level of input DNA in order to achieve complete cleavage.


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Acknowledgments We thank Elo Leung, Xiangdong Meng, Sarah Hinkley, and Lei Zhang for help with the design and assembly of ZFNs; Jianbin Wang and Geoff Friedman for transfections; and Philip Gregory, Susan Abrahamson, and Lei Zhang for helpful comments on the manuscript. References 1. Perez, E.E., Wang, J., Miller, J.C., et al. (2008) Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol. 26, 808–816. 2. Cai, C.Q., Doyon, Y., Ainley, W.M., et al. (2009) Targeted transgene integration in plant cells using designed zinc finger nucleases. Plant Mol Biol. 69, 699–709. 3. Doyon, Y., McCammon, J.M., Miller, J.C., et al. (2008) Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol. 26, 702–708. 4. Meng, X., Noyes, M.B., Zhu, L.J., Lawson, N.D., and Wolfe, S.A. (2008) Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol. 26, 695–701. 5. Moehle, E.A., Rock, J.M., Lee, Y.L., et al. (2007) Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc Natl Acad Sci USA. 104, 3055–3060. 6. Urnov, F.D., Miller, J.C., Lee, Y.L., et al. (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 435, 646–651. 7. Maeder, M.L., Thibodeau-Beganny, S., Osiak, A., et al. (2008) Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell. 31, 294–301. 8. Lombardo, A., Genovese, P., Beausejour, C.M., et al. (2007) Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol. 25, 1298–1306. 9. Bibikova, M., Golic, M., Golic, K.G., and Carroll, D. (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics. 161, 1169–1175. 10. Bibikova, M., Beumer, K., Trautman, J.K., and Carroll, D. (2003) Enhancing gene tar-

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