ZFNs were engineered that recognize SuR
loci using publicly available resources provided by the Zinc Finger Consortium.6,7
The Consortium-sponsored ZFN architecture uses two zinc finger arrays (ZFAs), each with three zinc fingers that collectively recognize a nine bp target site (Supplementary Fig. 1A
). The ZFAs are fused to a Fok
I nuclease domain, and a 5-7 bp spacer separates the target sites for the two arrays, allowing the nuclease to dimerize and cleave within the spacer. ZFA engineering is most robust for G-rich sequences,8
and four such target sites were selected in SuRB
for constructing ZFAs by modular assembly, namely the joining together of individual zinc fingers with predetermined specificities (sites 815, 1071, 1853 and 1947) (, Supplementary Table 1
. Thirty-two ZFAs were constructed, and electrophoretic mobility shift assays identified three arrays with DNA binding activity, two of which bind half sites for the 815 target. This low success rate is consistent with previous findings that ZFAs constructed by modular assembly are often non-functional.8
Figure 1 The tobacco SuRB locus. a) The diagram is drawn to scale and annotated with ZFN sites, amino substitutions that confer herbicide resistance, PCR primers used to characterize recombinants, and the region used as a donor template. b) ZFN target sites. Left (more ...)
Oligomerized Pool Engineering (OPEN) - a method developed by the Consortium - employs genetic selections in bacteria to identify ZFA variants that recognize specific target sequences6
(Supplementary Fig. 1B
). ZFAs made by OPEN typically show higher activity than those made by modular assembly, likely because the process of selection accommodates context-dependent interactions among neighboring zinc fingers in the array.6,10,11
OPEN was used to generate ZFNs for four sites (sites 865, 1853, 1947, 2163), including two that had been targeted by modular assembly (Supplementary Table 2
). Functional left and right ZFAs were obtained for the 1853 target, for which modular assembly had failed, as well as for target 2163. The OPEN-derived ZFAs showed activity in bacterial two-hybrid (B2H) assays,7
in which binding of ZFAs upstream of a lacZ
reporter gene activates expression (Supplementary Table 2
To test whether the ZFAs function as ZFNs, an assay was developed that measures ZFN activity in yeast (Supplementary Fig. 2A
). This assay uses a lacZ
reporter gene with a 125 bp internal DNA sequence duplication. The ZFN target site is cloned between the duplicated sequences, and cleavage of the target site creates a functional lacZ
gene through repair of the break by single strand annealing. ZFN activity is assessed by quantitative measurements of β-galactosidase activity. The six ZFAs for the 815, 1853 and 2163 target sites functioned effectively as ZFNs (). The 815 left and 1853 right arrays showed the most activity, comparable to activity observed with a ZFN designed from the well-characterized Zif268 ZFA.2
Figure 2 Activity of engineered ZFAs and ZFNs. a) ZFAs as stimulators of recombination in yeast. Target sites for each ZFA are listed in vertical text below the chart; H, high-copy plasmid; L, low-copy plasmid. Error bars denote s.d.; n = 3. b) Engineered ZFNs (more ...)
ZFNs were tested against their endogenous targets in tobacco by measuring whether they create mutations by non-homologous end-joining (NHEJ) (Supplementary Fig. 2B
). ZFN-encoding constructs were electroporated into tobacco protoplasts, and the relevant target sites in SuRA
were amplified by PCR and subjected to high-throughput pyrosequencing.12
The fraction of unique sequence reads showing size-polymorphisms (consistent with imprecise repair by NHEJ) was normalized to controls (, Supplementary Table 3
). Mutation frequencies were significantly higher for ZFN 815 at both SuR
loci. Interestingly, the highest mutation frequencies were not at the intended target in SuRB
, but rather at the corresponding sequence in SuRA
, which differs by two nucleotides. Not only does the ZFN 815 (which was created by modular assembly) lack specificity, but the higher level of mutagenesis at SuRA
relative to SurB
suggests other factors such as chromatin or DNA methylation influence access of this enzyme to target sites. In contrast to ZFN 815, the OPEN-designed ZFN 1853 only showed enhanced mutagenesis at its intended SuRB
target, which differs in sequence from the SuRA
site by a single nucleotide. This suggests that the genetic selections used in OPEN yield ZFNs with high specificity. No enhancement of mutagenesis was observed with ZFN 2163.
To measure whether the engineered ZFNs could stimulate incorporation of specific DNA sequence changes at SuR
loci by homologous recombination (HR) (Supplementary Fig. 2B
), three donor templates were constructed, each with a missense mutation that confers resistance to one or more herbicides (P191A, chlorsulfuron; S647T, imazaquin; W568L, chlorsulfuron and imazaquin)5,13
(). Silent nucleotide changes were introduced into codons adjacent to each mutation to distinguish the donor template from the native locus and spontaneous mutants from those generated by recombination (). An additional set of donor templates was made in which the ZFN recognition sites were altered to prevent cleavage ().
To test for gene targeting by HR, plasmids encoding the 815 ZFNs were electroporated into tobacco protoplasts with donor templates bearing the P191A, W568L or S647T mutations (, rows 1-3). The mean ZFN-induced herbicide resistance ranged from 5.3% for the P191A donor to 2.4% for the S647T donor. Both SuRA
were PCR-amplified from 12 randomly selected resistant calli derived from each treatment, using primers specific for the target locus (). DNA sequence analysis revealed that in 9 of 12 lines generated with both the P191A and W568L donor template, resistance was due to HR (P191A: 5 in SuRA
and 4 in SuRB
; W568L: 4 at SuRA
, 5 at SuRB
) (, rows 1-3, Supplementary Table 4
). With the S647T donor template, only 1 of the 12 herbicide resistant lines had evidence of HR (at SuRA
) and nine were spontaneous SuRB
mutants. For 8 of the 36 resistant lines in the gene targeting experiments, no mutations were observed in SuRA
, and so the molecular basis for the resistance is unknown. This resistance could be due to genotypic and phenotypic variation (somoclonal variation) typically observed when plant cells are grown in culture.14
Based on the number of recombinants recovered, the estimated gene targeting frequencies range from 4.0% for P191 to 0.2% for S647 ().
Gene targeting frequencies at SuRA and SuRB.
Molecular basis for herbicide resistance in gene targeting experiments.
One surprising outcome of the above experiment was that gene targeting frequencies exceeding 2% were obtained at a distance more than 1.3 kb from the cleavage site. This suggests that plant genes can be modified even when DNA sequence composition precludes engineering ZFNs near the desired site of modification. The high frequencies of recombination observed at both SuRA
with ZFN 815 are consistent with the pyrosequencing data indicating that this enzyme cuts promiscuously at both targets (). SuRA
differ at the nucleotide sequence level by 4%,5
and it is notable that high efficiency gene targeting could be achieved at SuRA
using the SuRB
-derived donor template.
We next tested the ability of the 1853 and 2163 ZFNs to stimulate HR and incorporate amino acid sequence changes near their respective target sites. Donor templates were used with mutations in the ZFN target site that prevent cleavage. ZFN 815 was used as a control, and the mutated donor did not substantially alter the overall frequency of herbicide resistance or gene targeting (, compare rows 1 & 4). The mutated P191A donor template did, however, cause an increase in the proportion of gene targeting events at SuRB
relative to SuRA
(, compare rows 1 & 4). Why inability to cleave the donor template influences the outcome of recombination is unclear. For the 1853 ZFN, the mean number of herbicide resistant events at W568L (281 bp from the cut site) was 0.6% (, row 5), more than 5-fold lower than gene targeting observed with ZFN 815 at much greater distances from the cut site. The 2163 ZFN yielded only three, non-targeted herbicide resistant calli in twelve separate experiments, two of which had mutations at sites in SuRB
previously known to confer herbicide resistance13
(Supplementary Table 5
and data not shown). The activity of all three ZFNs in HR parallels activities of these enzymes in the yeast and NHEJ assays (). Among the 47 herbicide resistant calli analyzed in the various gene targeting experiments, 19 (40.4%) showed modifications at multiple SuR
loci, including mutations introduced by NHEJ at the ZFN cleavage site (Supplementary Tables 4
). This indicates that transformed cells that sustain a ZFN-induced modification often incur changes at multiple alleles. Ten plants were regenerated from herbicide resistant calli and shown to carry the SuRA
mutations (Supplementary Tables 4
), indicating that ZFN-assisted gene targeting can be used to engineer genetically modified whole plants.
Based on the high frequency of gene targeting observed at SuRA
, we reasoned that populations of cells transformed with ZFNs might be screened directly for gene targeting events. To test this, protoplasts were transformed with plasmids encoding the 815 ZFN, the P191A donor template, as well as a neomycin phosphotransferase II (NPTII) gene that confers resistance to kanamycin. In this experiment, NPTII was used merely to identify cells that had been transformed. Approximately 1000 transformed cells were transferred to media with herbicide, and two calli displayed herbicide resistance, both of which carried mutations introduced by the donor template (Supplementary Table 4
). Although this experiment identified HR events at the SuR
loci by examining herbicide resistance, its success suggests that screens, perhaps using high throughput DNA sequencing, could be used to identify recombinants among populations of transformants for any genetic modification introduced by recombination, regardless of whether a selection exists for its associated phenotype.
Current methods for modifying plant genomes are limited to decades-old methods of DNA transformation that lack precision and control over the outcome of the modified chromosome. Plant biologists have long sought a method to make directed mutations in plant genes with high efficiency, as evidenced here with the use of ZFNs. Gene targeting offers numerous opportunities for studying plant gene function, and it also enables biosynthetic pathways to be harnessed to better produce much-needed plant-derived products. The ability to engineer highly functional ZFNs using publicly available reagents and to recover HR-induced mutations even at considerable distances from the ZFN cleavage site demonstrate that targeted mutagenesis in plants is now practical.