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. 2011 Jun;156(2):466-73.
doi: 10.1104/pp.111.172981. Epub 2011 Apr 4.

Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases

Shaun J Curtin  1 , Feng Zhang, Jeffry D Sander, William J Haun, Colby Starker, Nicholas J Baltes, Deepak Reyon, Elizabeth J Dahlborg, Mathew J Goodwin, Andrew P Coffman, Drena Dobbs, J Keith Joung, Daniel F Voytas, Robert M Stupar
Affiliations

Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases

Shaun J Curtin et al. Plant Physiol. 2011 Jun.

Abstract

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform--a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting dicer-like (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome.

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Figures

Figure 1.
Figure 1.
Detection of ZFN-induced mutations at a GFP transgene in soybean. A, The position of the OPEN ZFN target site is represented by a gray rectangle. The target sequence of both left and right ZFAs recognize a 9-bp sequence (indicated in bold). A strategy involving the restriction enzyme BccI and a PCR assay was used to enrich and identify mutated sequences. B, Amplicons from the PCR assay were cloned into pGem T-easy and subsequently amplified by colony PCR using the GFP-specific primers (Supplemental Table S3). The sequencing of PCR products revealed large deletions ranging from 27 to 71 bp. The enrichment of mutated GFP sequence was biased toward large deletions at the 5′ region of the target site since the BccI recognition site CCATC was located on the left ZFA recognition sequence. Typically the restriction site is situated in the middle of the target site, as the majority of obtained indels are minor (1–10 bp) and occur in the spacer region.
Figure 2.
Figure 2.
Detection of ZFN-induced mutations in soybean hairy-root tissue. A, A schematic strategy highlighting the restriction endonuclease PCR assays used to enrich mutated DNA sequences from soybean hairy-root tissue. Five ZFNs were designed to target seven genes, two of which targeted duplicate copies of DCL1 and DCL4 (DCL1a and DCL1b were successfully targeted by one ZFN construct, but are shown in different sections because they were screened with paralog-specific primers; DCL4a and DCL4b were successfully targeted by one ZFN construct and are shown together because they were screened with a shared primer set). A single ZFN capable of targeting both copies of RDR6 could not be identified. The closest match for both RDR6a and RDR6b was a target site that differed by 2 bp. Two different ZFN constructs were designed to target these respective sites. A ZFN was designed to target one of the duplicate copies of HEN1. B, The PCR products from four root samples, including an undigested (WT/U) and digested (WT/D) wild-type control, were separated on a 2% agarose gel. Lanes showing undigested products indicate a portion of the hairy-root cells having novel mutations at the restriction sites.
Figure 3.
Figure 3.
Sequences of induced ZFN mutations in soybean hairy-root tissue. The recovered mutated alleles from seven soybean endogenous genes are shown below their respective wild-type sequence. The bold and underlined sequences represent the ZFN target sites of each wild type. Deletions and insertions are indicated by dashes or lowercase letters, respectively. Single roots often produced multiple independent mutations. The numbers to the side indicate the type of mutation and how many nucleotides were involved.
Figure 4.
Figure 4.
The mutagenic specificity of the RDR6a and RDR6b ZFN transgenes was assessed by performing PCR enrichment assays with gene-specific primers for each homeolog. The ZFN target sites of this gene pair differ by only two nucleotides, so this experiment was important to measure whether the gene-specific ZFNs could discriminate between the homeologous targets (Table I provides details on the target site differences of the two ZFN transgenes). The PCR enrichment assays are analogous to those shown in Figure 2. A sample of estradiol-induced hairy roots was targeted for mutagenesis using the ZFN transgene targeting RDR6a (roots 1–4) and the ZFN transgene targeting RDR6b (roots 5–8). Primer sets differing at a single nucleotide were designed to allow for homeolog-specific PCR amplification in these samples (the polymorphic nucleotide is underlined in the reverse primer sequences). The top section shows the PCR enrichment results when testing for mutagenesis of gene RDR6a and the bottom section shows the PCR enrichment results when testing for mutagenesis of gene RDR6b. In either case, an undigested top band indicates a mutation within the hairy-root sample. A digested nontransgenic root sample (WT/D) and an undigested nontransgenic root sample (WT/U) serve as controls. Seven out of the eight targeted hairy roots show the presence of the putative mutated top band; root 4 does not show this band, indicating that this sample either failed to transform or the ZFN failed to mutagenize any cells in this root. Faint top band shadows are observed in some samples for the nontargeted gene. Therefore, we cannot rule out the possibility that some mutations may have occurred at the nontargeted homeologous gene, albeit at a much lower frequency than the targeted gene.
Figure 5.
Figure 5.
ZFN mutagenesis and heritability in whole-plant soybean. A, Genomic structure of the DCL4b gene in soybean. The target site is highlighted by dashed lines with the box indicating the ZFN-induced dcl4b mutation (2-bp insertion indicated in bold) relative to the wild type. B, A schematic of the strategy used to determine the segregation frequency of the homozygous and heterozygous mutations in the T1 progeny. C, A gel depicting the segregation of the mutation in 14 T1 plants is shown (PCR results for the remaining 10 plants are not shown). The top section shows the DCL4b genotype (+/+ indicates DCL4b/DCL4b, −/− indicates dcl4b/dcl4b, and +/− indicates heterozygous DCL4b/dcl4b). The middle section shows the genotyping result for the ZFN transgene (BAR amplicon) and the bottom section shows the PCR positive control (Actin amplicon). The induced dcl4b mutation segregated as expected in the 1:2:1 ratio. PCR confirmed that all T1 plants, with the exception of the wild-type control and one heterozygous plant (lane nine), harbored the ZFN transgene.
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