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Molecular and Cellular Biology, January 2001, p. 289-297, Vol. 21, No. 1
Department of Biochemistry, University of
Utah School of Medicine, Salt Lake City, Utah
84132,1 and Department of
Environmental Health Sciences, The Johns Hopkins University School
of Hygiene and Public Health,2 and
Department of Biophysics and Biophysical Chemistry, The
Johns Hopkins University School of Medicine,3
Baltimore, Maryland 21205
Received 28 August 2000/Returned for modification 2 October
2000/Accepted 5 October 2000
Chimeric nucleases that are hybrids between a nonspecific DNA
cleavage domain and a zinc finger DNA recognition domain were tested
for their ability to find and cleave their target sites in living
cells. Both engineered DNA substrates and the nucleases were
injected into Xenopus laevis oocyte nuclei, in which
DNA cleavage and subsequent homologous recombination were
observed. Specific cleavage required two inverted copies of the zinc
finger recognition site in close proximity, reflecting the need for
dimerization of the cleavage domain. Cleaved DNA molecules were
activated for homologous recombination; in optimum conditions,
essentially 100% of the substrate recombined, even though the DNA was
assembled into chromatin. The original nuclease has an 18-amino-acid
linker between the zinc finger and cleavage domains, and this enzyme cleaved in oocytes at paired sites separated by spacers in the range of
6 to 18 bp, with a rather sharp optimum at 8 bp. By shortening the
linker, we found that the range of effective site separations could be
narrowed significantly. With no intentional linker between the binding
and cleavage domains, only binding sites exactly 6 bp apart
supported efficient cleavage in oocytes. We also showed that two
chimeric enzymes with different binding specificities could collaborate
to stimulate recombination when their individual sites were
appropriately placed. Because the recognition specificity of zinc
fingers can be altered experimentally, this approach holds great
promise for inducing targeted recombination in a variety of organisms.
Procedures and reagents that allow
the directed alteration of genes in situ constitute a powerful toolbox
for experimental genetics and potentially for agricultural and
therapeutic applications. In many organisms, however, and particularly
in higher eukaryotes, the efficiency of recombination between an
introduced DNA and the homologous chromosomal target is discouragingly
low. For example, such events typically occur in mammalian cells at a
frequency of only about 1 for each 106 cells treated
(3, 31). We are interested in developing procedures that
would substantially improve the frequency of gene targeting.
A major impediment to efficient gene replacement is the status of the
chromosomal target. Increasing the number of target sequences has
little or no effect on targeting efficiency (54, 60). In
contrast, making an intentional double-strand break (DSB) in the target
DNA increases the yield of specific homologous recombination events up
to 1,000-fold or more (10, 11, 14, 44, 46). It is believed
that exonucleases act at broken ends to generate single-stranded tails
that are recombinagenic in any of several pathways. In particular, the
single-strand annealing mechanism (33), by which
homologous recombination involving exogenous DNA usually occurs in
higher eukaryotes (53), cannot proceed unless both the
donor and target have ends (5, 48).
Whatever the mechanism of recombination, it is clear that the frequency
of targeted recombination can be substantially improved by introducing
a targeted DSB. The feasibility of this approach has been demonstrated
by directing cleavage with meganucleases, like I-SceI
(20); however, the utility of such enzymes is limited by
the need to introduce the corresponding recognition site by a
traditional, low-efficiency process before it can be cleaved. More
useful would be cleavage reagents that either inherently possess or can
be designed to have affinity for natural chromosomal sequences. If the
recognition specificity of such reagents could be manipulated to attack
different targets in different circumstances, this would be most powerful.
In the present study we investigate the potential of a class of
chimeric nucleases for DNA cleavage and initiation of recombination in
living cells. These enzymes are hybrids between the nonspecific cleavage domain of the type IIs restriction endonuclease
FokI and a DNA-binding domain made up of three
Cys2His2 zinc fingers (Fig.
1A) (7, 17, 24, 26-30, 37,
51). Recognition of DNA by zinc fingers is modular: each finger
contacts primarily three consecutive base pairs in the target, and a
few key residues in the protein mediate recognition. These features
have encouraged attempts to develop zinc finger combinations with novel
specificities, which have proved quite successful (8, 12, 13, 16,
18, 19, 43, 49, 58, 59). In fact, randomization of the codons for the recognition residues allows the selection of new fingers that
have high affinity for arbitrarily chosen DNA sequences. Furthermore,
zinc fingers are natural DNA-binding modules, and engineered fingers
have been shown to act on their designed targets in living cells
(1, 9, 25, 34). Thus, nucleases based on zinc fingers
should be targetable to specific but arbitrary recognition sites.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.289-297.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Stimulation of Homologous Recombination through
Targeted Cleavage by Chimeric Nucleases
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (16K):
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FIG. 1.
(A) Schematic diagram of a chimeric nuclease and DNA
substrate. The nuclease consists of three zinc fingers (Zn) connected
to the cleavage domain of FokI (FN) by a
flexible peptide linker. The N and C termini of the protein are
indicated. Each finger makes contact with three consecutive base pairs
in the recognition sequence. In the DNA substrates, the canonical
binding site for QQR, 5'-GGG GAA GAA, was inserted, in
various numbers and orientations, between the 1.25-kb direct repeats
(boxes with arrows) of plasmid pRW4. (B) Scheme for the oocyte
injection experiments. The DNA substrate is diagrammed at the top left,
and the position of the unique PvuII site is shown. For each
sample the DNA was injected into the nuclei of 20 to 40 oocytes; they
were incubated for 3 to 4 h to allow chromatin assembly, and then
QQR was injected into the nuclei. After various lengths of time, DNA
was recovered from the oocytes, digested with PvuII, and
analyzed by Southern blot hybridization. This distinguishes DNA
molecules not cleaved by QQR (Uncut) from cleaved DNA (Cut) and from
cleaved molecules that have undergone homologous recombination (Rec).
The locations of the PvuII sites and the sizes of the
resulting PvuII fragments are shown.
Here we characterize the cleavage abilities of the chimeric nucleases in Xenopus laevis oocytes. These enormous cells have a large capacity for homologous recombination that is readily accessed by microinjection of appropriate substrates (5) and that proceeds by the same single-strand annealing mechanism that is the principal pathway available to exogenous DNAs in cultured mammalian cells (5, 53). Injected linear DNAs undergo efficient recombination if they carry appropriately placed homologous sequences, and a single oocyte can process more than 109 molecules into completed recombination products in a few hours (6, 35, 36). Injected circular DNAs are assembled into apparently normal chromatin and are inert for recombination, but they can be induced to interact with a homologous partner, if they are cleaved (48). A circular DNA thus serves as an effective model for an inactive chromosomal target.
We report that chimeric nucleases based on zinc fingers are capable of finding their recognition sites in oocytes, directing specific cleavage, and stimulating local homologous recombination. The substrate requirements for cleavage in living cells are described, and future applications are discussed.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Enzymes.
Zif-QQR-FN (29) and
Zif-
QNK-FN (51) were purified from
overproducing bacteria as previously described (51, 52).
We refer to them by the three-letter designations QQR and QNK,
respectively. Coding sequences for enzyme variants with altered linkers
were created from the original QQR clone in the pET15b vector by PCR using primers carrying the desired alterations. The resulting plasmids
were verified by sequence analysis and transformed into Escherichia coli BLR(DE3)/pLysS; induction, lysis, and
enrichment of the enzymes were carried out as described for QQR
(51, 52). In vitro reactions were performed in 20 µl
containing 10 mM Tris (pH 8.5), 50 mM NaCl, 1 mM dithiothreitol, 100 µM ZnCl2, 50 µg of bovine serum albumin per ml, 100 µg of tRNA per ml, and 50 ng of substrate DNA (final concentration,
about 0.7 nM). After addition of various amounts of enzyme, the mixture
was incubated for 30 min at room temperature. MgCl2 was
added to a final concentration of 10 mM, and incubation was continued
for 1 h at room temperature. Cleavage was monitored by
electrophoresis in 1% agarose gels.
DNA substrates. The parent plasmid, pRW4, consists of pBR322 sequences with a direct duplication of 1.25 kb and a unique XhoI site between the repeats (36). Substrate DNAs were constructed by insertion of oligonucleotide duplexes containing the recognition site for QQR (5'-GGG GAA GAA) and/or QNK (5'-GGG GCG GAA), in various arrangements, at the XhoI site. Sequences of the inserts were verified experimentally in all cases, and they are reported explicitly elsewhere (52).
Oocyte injections. Injections, DNA recovery, and analysis were performed as described previously (4, 36). A mixture containing 1 ng of substrate DNA (about 0.3 fmol) and 1 ng of the recovery control plasmid pHSS6 (22) was injected into each oocyte in a volume of 20 nl. After incubation for 3 to 4 h to allow chromatin assembly, the chimeric nuclease in 10% glycerol was delivered to the nuclei in a volume of 2.5 to 15 nl. Two different QQR solutions were used for injections: one with an estimated concentration of 3 fmol/nl, and the other with 7 fmol/nl. The linker variant enzymes were approximately 7 fmol/nl, and the QNK stock was approximately 3 fmol/nl. Because glycerol can be toxic to oocytes, the injection volume was limited. Some aliquots of enzyme tended to lose activity in the injection needles, so care was taken to load a fresh needle for each small batch of oocytes and to use it within about 20 min. Samples taken from the needle before and after oocyte injections were assayed to ensure that the enzyme remained active, and only examples that retained activity were processed further.
After incubation and recovery of the DNA, it was digested with PvuII and analyzed by Southern blot hybridization with radioactive pBR322 as a probe. The radioactivity in each band was quantitated with a Molecular Dynamics model 400E PhosphorImager using ImageQuant software. Reported percentages of recombination product were calculated as R/(R + U) × 100, where R is the number of counts in the recombinant band and U is the counts in the uncut substrate. The intensity of the pHSS6 control band (C) was used to determine the recovery of substrate DNA by comparing (R + U)/C in the recovered DNA to U/C in an uninjected sample. While there appeared to be some loss of cleaved DNA in some cases, the total recovery was essentially always above 80%.| |
RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Experimental design. The general designs of the enzymes and DNA substrates used in this study are shown in Fig. 1A. The chimeric nucleases are composed of three zinc fingers at the N terminus, connected by a flexible linker to the cleavage domain of FokI (FN) at the C terminus. Each finger contacts primarily three consecutive base pairs of DNA. Each plasmid DNA carries one or more copies of the recognition sequence for the chimeric nuclease between 1.25-kb direct repeats.
The protocol for injection into Xenopus oocytes is illustrated in Fig. 1B. For each combination of experimental parameters, the substrate DNA was injected into the nuclei of 20 to 40 oocytes and allowed 3 to 4 h to assemble into chromatin (15, 48). We have verified chromatin assembly in our conditions by showing that only about one-quarter of restriction sites in the plasmids are susceptible to cleavage by enzymes injected into the oocytes and that recovered DNA is supercoiled after deproteinization (data not shown). The chimeric nuclease was then injected, again directly into the oocyte nucleus. DNA was recovered after various times of incubation and assayed for cleavage and recombination by digestion with PvuII, gel electrophoresis, and Southern blot hybridization. Substrate molecules that were not cut by the chimeric nuclease, cut molecules, and molecules that had undergone homologous recombination all yield characteristic fragments in this analysis (Fig. 1B). Although recombination products may be formed from these substrates largely by intramolecular events, we have shown previously that intermolecular reactions, very similar in principle to a gene-targeting setup, are stimulated to a comparable extent by directed DSBs in oocytes (5, 48).Requirements for cleavage and recombination in oocytes.
Figure
2A shows a time course experiment in
which a plasmid DNA with paired inverted recognition sites for QQR
(pQT10) was injected into oocytes and then isolated at various times
after enzyme injection. Products of cleavage at the expected site (Cut) were observed within 1 h after enzyme injection. There are two cut
bands because the linear DNA was digested with PvuII for
analysis. A band corresponding to recombination products (Rec) was also visible, and in longer exposures a faint trailing smear representing recombination intermediates (35) was seen. Both cleavage
and recombination proceeded through the 3-h time point, and the process was essentially complete by 6 h. This corresponds well to time courses of recombination of linear DNAs in oocytes determined previously (5, 35). In this experiment, the final level of recombination was 54%; both cleavage and recombination products were
absent if the nuclease was not injected (6, 48) (data not
shown). The total recovery of injected substrates was always good, as
indicated by comparison to the circular recovery control plasmid pHSS6.
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Influence of recognition site separation on efficiency.
A
series of substrates carrying paired inverted repeats of the
recognition site separated by various lengths of spacer DNA were tested
for the ability of QQR to stimulate recombination in Xenopus
oocytes. Two different experiments are shown in Fig. 3, one that includes a broad range of
site separations (4 to 35 bp; Fig. 3A), and the other that concentrates
on the range between 12 and 20 bp (Fig. 3B). Effective cleavage and
recombination occurred with several substrates, but there was a rather
sharp dependence on the length of the spacer. Little product was formed
when the separation was 4 bp; this substrate was also not cleaved
effectively in vitro, probably due to steric clash between enzyme
molecules that attempt to bind to the two sites (52). The
yield was better with a separation of 6 bp, and a spacer of 8 bp
consistently gave the largest yield. In the experiment shown in Fig.
3A, 94% of this substrate was converted to recombination product; in
Fig. 3B the yield was 95%. A secondary maximum was often seen with a
16-bp spacer (Fig. 3B). The efficiency dropped at larger separations, and with distances of 20 bp and greater, very little or no product was
formed. (In a separate experiment [not shown], a separation of 5 bp
gave no recombination, and the yield with 7 bp was intermediate between
those for 6 and 8 bp.)
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Linker variants in vitro.
The specificity of the chimeric
nuclease is determined by how many related sites are recognized and
cleaved. This is governed largely by the discrimination achieved by the
zinc fingers, but it also reflects how many different configurations of
the binding sites can be effectively recognized. As illustrated in Fig.
4, when the binding sites are moved
farther apart, the linker must extend a longer distance. For a given
linker length, there will be a limit to the distance between
recognition sites that is consistent with both binding and
dimerization. When the linker is shortened, we expect this limit to be
reduced. We therefore attempted to limit the range of competent targets
by shortening the flexible peptide linker between the binding and
cleavage domains of QQR.
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3, and L6, respectively. These enzymes were used to treat the range of substrates shown in Fig.
3 both in vitro and in oocytes. (L6 was not studied in oocytes due to
the relative ineffectiveness of L3.)
As noted earlier, L18 is capable in vitro of cleaving to completion all
substrates with 6 to 35 bp between recognition sites, while spacers of
4, 5, and 40 bp do not support cleavage. Examining the altered enzymes,
we found a progressive decrease in cleavage of some DNAs with
decreasing linker length (Table 1). The
first to show reduced cleavage were spacers of 10, 20, and 30 bp, which were essentially resistant to L6 and all smaller linkers. Next, distances of 8, 18, and 35 became resistant, and finally cleavage of
the 7- and 12-bp spacers disappeared. Concomitantly, DNAs with spacers
of 4 and 5 bp became sensitive as the linker was shortened. The 5-bp
construct was cleaved moderately well by L6 and very effectively by all
shorter linkers, while the 4-bp target showed weak cleavage by L2 and
L0 and strong cleavage by L3 and L6.
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3 and L6).
Linker variants in oocytes.
We tested the capabilities of the
same enzyme variants (except L6) to cut substrates with different
spacer lengths in oocytes. The basic observations were that the range
of susceptible targets became restricted as the linker was shortened;
and for each enzyme, as with QQR (L18), the range was narrower in cells
than in vitro. Some examples are shown for L0 in Fig.
5. At moderate concentrations of the
nuclease, essentially the only substrate cleaved was that with a 6-bp
spacer. Neither the 5-bp nor the 7-bp construct showed any
recombination, and those with 14-bp and 16-bp spacers gave very weak
product bands. At higher enzyme inputs (not shown), greater cleavage of
the 14- and 16-bp substrates could be forced, but there was still no
cleavage of any shorter spacer except 6 bp.
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Cleavage of paired nonidentical recognition sites. When cleavage of a chromosomal target is desired, it is very unlikely that exact inverted repeats of a 9-bp sequence will be located in favorable positions. Thus, it will be necessary to devise nucleases with two different sets of zinc fingers designed to bind two different 9-mers. We tested the feasibility of this scheme using two chimeric nucleases that recognize related but different sites. The preferred site for QQR is 5'-GGG GAA GAA (29, 50), while that for QNK is 5'-GGG GCG GAA (23, 51). Three substrates were prepared: one with two sites for QQR (QQ), one with two sites for QNK (KK), and one with one site of each type (QK). An 8-bp spacer was chosen because it was optimal for cleavage and recombination in oocytes (see Fig. 3). Earlier experiments had shown that similar substrates with 14-bp spacers were cleaved in vitro by a combination of the two enzymes (52).
When the hybrid substrate was injected into Xenopus oocytes, it was activated effectively for recombination only when both enzymes were injected together (Fig. 7). Both single-enzyme controls were positive on their own substrates, but cross-cleavage was not observed. Injection of QQR stimulated recombination of the QQ substrate, and the yield of product was 29%. Injection of QNK led to cleavage and recombination of KK, with a yield of 16%. With the combined substrate (QK), essentially no recombination was observed when QQR was injected alone; with QNK a low level of recombination was seen (yield, 2%), reflecting the lower selectivity of this enzyme. When a mixture of the two enzymes was injected, a level of recombination was observed (yield, 19%) that was comparable to that obtained with the single-enzyme substrates. Thus, two enzymes directed to nonidentical sites can collaborate to produce a recombinagenic DSB in cells.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Requirements of the chimeric nucleases. Our long-term goal is to provide reagents that will substantially improve the frequency of targeted homologous recombination. Because DSBs stimulate recombination dramatically in their vicinity in a wide variety of organisms (10, 11, 14, 38, 39, 41, 44, 46-48), we have focused our attention on chimeric nucleases that have the potential for making targeted breaks at arbitrarily selected DNA sequences.
The enzymes of particular interest carry a nonspecific DNA cleavage domain linked to a DNA-binding domain comprised of Cys2His2 zinc fingers. We have shown that these chimeras are capable of locating their target sequences in chromatin, cleaving with good efficiency, and thereby stimulating homologous recombination. Although the substrates we used were engineered plasmid DNAs and the cellular milieu was that of the Xenopus oocyte, our findings should be applicable to mammalian somatic cells and to many other cells and organisms that do not have the ability to initiate recombination efficiently at unbroken chromosomal targets. This might include organisms popular with geneticists, like Drosophila melanogaster and nematodes. In the former case, target cleavage might be combined with the recently described procedures for producing linear donor DNA in vivo (45) to achieve maximum efficiency. Our results define the requirements for cleavage by the chimeric nucleases in living cells. Because dimerization of the cleavage domain is required for nuclease activity (2, 52), two recognition sites for the zinc fingers must occur in close proximity. These sites must be in the inverted repeat orientation that directs the two cleavage domains toward the space between the sites. Furthermore, only a limited range of separations between binding sites is tolerated. With the original 18-amino-acid linker between the binding and cleavage domains of QQR, the range was 6 to 18 bp. When the linker was shortened, the range of effective spacer lengths was correspondingly constrained; and with no intentional linker between the domains, a separation of 6 bp was the only one cleaved efficiently. When the above criteria were met and sufficient enzyme was provided, essentially all of the substrate molecules were cleaved in oocytes. This indicates that assembly into chromatin does not prevent access of the nucleases to their targets. There is reason to be optimistic that this will be true with other chimeras based on zinc fingers, since these domains are derived from transcription factors that are capable of locating their recognition sequences in the normal nuclear environment. Furthermore, other engineered zinc fingers have been shown to modulate transcription at their specific targets in mammalian cells (1, 9, 25, 34).Interpretation of linker variants. Our earlier molecular modeling of the complexes of the chimeric nucleases with DNA (52) provides a basis for evaluating the results with interdomain linkers of different lengths (see Fig. 4). Several conclusions were drawn. First, it was certainly anticipated that the upper limit on spacers that allow effective cleavage would be reduced as the linker was trimmed, since the linker must extend to allow dimerization of the cleavage domains attached to the two separately bound recognition domains.
Second, short spacers that did not allow cleavage by the original QQR (L18) were cleaved when the linker was reduced. Although the linker does not form part of the interface that causes steric interference between binding and cleavage domains, shortening it may allow adjustments in other parts of the cleavage domains to allow closer approach of the binding domains. Third, the secondary maximum in cleavage efficiency at separations of 14 to 16 bp reflects the helical nature of DNA. This phenomenon is observed both in vitro (Table 1) and in oocytes (Fig. 3 and 6). The modeling and cleavage mapping results show that when the spacer between sites is 16 bp, the cleavage dimer sits off-center, 3 bp from one site and 13 bp from the other, and both linkers lie on the same helical face (52). When the separation between sites is 8, 10, or 12 bp, all possible domain arrangements require the linker to traverse around the helix to some extent. This extends the distance the linker must stretch, and it also seems that there is some inherent preference to keep the linker on one side of the DNA (52). Fourth, it is still puzzling that enzymes with very short linkers cleave substrates with sites separated as widely as we observed. The 6-bp spacer requires the linker to stretch 18 to 20 Å by our estimate (52) (Fig. 4). In the L0 construct, there are three residues in each binding domain and three in each cleavage domain that are disordered in the crystal structures; these six residues could easily reach the necessary distance. This same enzyme also cuts the 16-bp substrate efficiently, however, and the required extension in that case is about 40 Å for one of the linkers. Furthermore, the L3
and L6 constructs, in which more of the cleavage domain has been
deleted, also cut both the 6-bp and 16-bp substrates in vitro. It
appears that some of the cleavage domain must become unstructured in
order to accommodate these distances. The first regular feature at the
N terminus of the cleavage domain is an 
-helix that may be
stabilized in native FokI by interaction with elements of
the binding domain (55, 56). In the chimeric nucleases, it
is conceivable that this helix may be unfolded with modest energy
input. In addition, the actual distance between the binding and
cleavage domains could be reduced by a change in the DNA structure, for
example, a bend or kink at the site of cleavage.
Cleavage parameters in oocytes.
Cleavage by the chimeric
nucleases in oocytes shows stricter limits on effective recognition
site separations with all linker deletions. While a site separation of
8 bp is optimal for the L18 enzyme, the optimum shifts to 6 bp as the
linker is trimmed. The secondary optimum with spacers of 14 and 16 bp
is maintained in oocytes except for the L3 enzyme, presumably
reflecting the preference for the linker to remain on one side of the
DNA. With the L0 nuclease there is a very strong preference for sites
exactly 6 bp apart, and this fact should enhance the specificity of cleavage.
Applications to gene targeting. A chromosomal gene targeting experiment utilizing the chimeric nucleases would presumably proceed as follows. A target site would be chosen within a gene of interest. Zinc finger combinations would be derived that bind inverted sites separated by 6 bp at the target locus. These sites need not be identical, as we have demonstrated that two chimeras with different DNA-binding domains are capable of collaborating to achieve cleavage. The zinc finger domains would be linked to the DNA cleavage domain and tested in vitro for specificity to ensure that the zinc fingers recognize the desired sites. For maximum specificity, linkerless (L0) constructs would be made; but if suitable sites spaced by exactly 6 bp could not be found, longer linkers could be incorporated to accommodate greater separations. The two new chimeric nucleases would then be delivered to cells along with a linear donor DNA molecule carrying the desired sequence alteration. The method of delivery would depend on the organism, cell type, and other experimental conditions.
Will the chimeric nucleases have sufficient specificity to attack the desired target without introducing breaks at many other chromosomal locations? The requirement for dimerization of the cleavage domain enforces a high level of sequence specificity, as long as the zinc fingers show good discrimination against related sites. Since each set of three fingers binds nine consecutive base pairs, two chimeric nucleases effectively demand an 18-bp target if each zinc finger domain has perfect specificity. Any given sequence of this length is predicted to be unique in a DNA as complex as the human genome (3 × 109 bp), since there are 418 (6.9 × 1010) different 18-mers. Furthermore, it has been shown that additional fingers provide enhanced specificity (1, 25, 34), so the number of zinc fingers in each DNA-binding domain could be increased. What are the prospects for deriving zinc finger combinations that can recognize any desired 9-bp sequence? Fingers with new binding specificities have been produced by randomizing coding sequences for key residues that contact DNA, then selecting by phage display for combinations that bind the desired target most avidly (8, 12, 13, 19, 43, 59). In compelling demonstrations of the power of this approach, Greisman and Pabo (16) evolved zinc fingers that recognized completely new 9-bp sites by selecting sequentially for one finger at a time, and Segal et al. (49) systematically derived fingers that recognize the complete subset of GNN triplets. It is not known what limitations might exist on the ability of zinc fingers to bind the full spectrum of possible target sequences, but it is clear that the accessible range is large (18, 58). Several additional issues remain to be addressed to confirm the utility of chimeric nucleases as tools for gene targeting. Among these are demonstrating discrimination against related sequences; proving the efficacy of zinc fingers designed to bind arbitrarily chosen sequences; and testing the cleavage of genuine chromosomal targets. The question of discrimination among potential binding sites is a particularly critical one. In this regard, neither QQR nor QNK is the ideal model enzyme, since both can bind alternative sites (23, 29, 51). Impressive zinc finger binding selectivity has been achieved recently with the assistance of negative selection against closely related base triplets (49). An additional concern is the existence of nonhomologous recombination pathways, which will compete with homologous recombination to repair the broken target. It may be possible to take advantage of differences in the genetic requirements of these processes (21, 32, 40) to tip the balance in favor of homologous events. Assuming that these issues can be resolved satisfactorily, the use of chimeric nucleases for targeted gene manipulation should be applicable to a wide variety of organisms and experimental purposes. At present the effort required to produce zinc finger combinations with novel binding specificities will likely restrict application of the chimeric nucleases to situations in which the same site is targeted repeatedly. As experience accumulates, methods of producing new specificities will be improved, and even single-use applications may become feasible.| |
ACKNOWLEDGMENTS |
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This project is an equal collaboration between the Carroll and Chandrasegaran labs.
We thank Frank Whitby for providing Fig. 4. We are grateful to Jeremy Berg for advice on zinc finger recognition, to H. O. Smith for continuing interest in this project, and to Tim Formosa, Wes Sundquist, and Mario Capecchi for comments on various versions of the manuscript.
This work was supported in part by grants from the National Institutes of Health (GM50739 and GM58504) and the University of Utah to D.C. and by grants from the National Institutes of Health (GM53923) and the National Science Foundation (MCB 9415861) to S.C. Assistance was also provided by the Markey Center for Protein Biophysics, the Huntsman Cancer Institute at the University of Utah, and the Environmental Health Sciences Core Facility at Johns Hopkins University. S.C. is a member of the Scientific Advisory Board of Sangamo Biosciences, Inc.
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FOOTNOTES |
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* Corresponding author. Mailing address for Dana Carroll: Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84132. Phone: (801) 581-5977. Fax: (801) 581-7959. E-mail: carroll{at}path.utah.edu. Mailing address for Srinivasan Chandrasegaran: Department of Environmental Health Sciences, The Johns Hopkins University School of Hygiene and Public Health, 615 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 614-2289. Fax: (410) 955-0617. E-mail: chandra{at}welchlink.welch.jhu.edu.
Present address: Department of Molecular Biology, The Scripps
Research Institute, La Jolla, CA 92037.
Present address: Department of Biology, Massachusetts Institute of
Technology, Cambridge, MA 02139.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Molecular and Cellular Biology, January 2001, p. 289-297, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.289-297.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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