High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases

Abstract

We report here an efficient method for targeted mutagenesis of Arabidopsis genes through regulated expression of zinc finger nucleases (ZFNs)-enzymes engineered to create DNA double-strand breaks at specific target loci. ZFNs recognizing the Arabidopsis ADH1 and TT4 genes were made by Oligomerized Pool ENgineering (OPEN)-a publicly available, selection-based platform that yields high quality zinc finger arrays. The ADH1 and TT4 ZFNs were placed under control of an estrogen-inducible promoter and introduced into Arabidopsis plants by floral-dip transformation. Primary transgenic Arabidopsis seedlings induced to express the ADH1 or TT4 ZFNs exhibited somatic mutation frequencies of 7% or 16%, respectively. The induced mutations were typically insertions or deletions (1-142 bp) that were localized at the ZFN cleavage site and likely derived from imprecise repair of chromosome breaks by nonhomologous end-joining. Mutations were transmitted to the next generation for 69% of primary transgenics expressing the ADH1 ZFNs and 33% of transgenics expressing the TT4 ZFNs. Furthermore, approximately 20% of the mutant-producing plants were homozygous for mutations at ADH1 or TT4, indicating that both alleles were disrupted. ADH1 and TT4 were chosen as targets for this study because of their selectable or screenable phenotypes (adh1, allyl alcohol resistance; tt4, lack of anthocyanins in the seed coat). However, the high frequency of observed ZFN-induced mutagenesis suggests that targeted mutations can readily be recovered by simply screening progeny of primary transgenic plants by PCR and DNA sequencing. Taken together, our results suggest that it should now be possible to obtain mutations in any Arabidopsis target gene regardless of its mutant phenotype.

Fig. 1.

OPEN ZFN target sites in the ADH1 and TT4 coding sequences. (A) The gene models of Arabidopsis ADH1 and TT4 loci are shown as block arrows. Black rectangles represent exons; white rectangles represent introns. The positions of OPEN ZFN target sites are indicated by triangles. Each engineered ZFA consists of three zinc fingers (F1, F2, F3), which together recognize a 9-bp target site. The two target sites are separated by 6-bp spacer sequences. Binding of the ZFAs to the target sequences enables the FokI nuclease monomers (indicated as gray ovals) to dimerize and cleave within the spacer. The sites of cleavage in the target sequences are indicated by black arrowheads. (B) ZFAs generated by OPEN were tested for their activity in bacterial two-hybrid (B2H) assays. The ZFAs were tested for their ability to bind target sequences in bacteria and activate transcription of a downstream lacZ reporter gene. Fold activation of each ZFA relative to its negative control, which does not express the ZFA, is plotted on the y axis. Multiple ZFAs generated by OPEN were tested for each of the ADH1 and TT4 left and right target sites. ZFAs with activities indicated by black bars were used in subsequent tests. Error bars denote SD; n = 3.

Fig. 2.

ZFN activity in yeast. (A) Schematic of the yeast-based ZFN activity assay. ZFN expression plasmids are introduced into a yeast strain of α mating-type; the reporter plasmid is transformed into a yeast strain of a mating type. Mating of the two strains brings together the ZFNs and the reporter plasmid into a diploid cell. This results in cleavage of the ZFN target sequence and restoration of the lacZ gene through single strand annealing (SSA). (B) Results of the yeast assay performed with the ADH1 and TT4 ZFNs. A ZFN derived from the well-characterized ZFA, Zif268, is included as the positive control. Negative controls are the ADH1 and TT4 target sites combined with the Zif268 ZFN. Error bars denote SD; n = 3.

Fig. 3.

Detection of ZFN-induced mutations in Arabidopsis protoplasts and somatic cells. (A) Schematic of the strategy used to amplify and identify mutations at the ZFN target sites (gray rectangle). Restriction enzyme sites coincident with the ZFN cleavage sites are NlaIII for ADH1 and NspI for TT4. Short black arrows indicate the positions of PCR primers relative to the target sites. (B) Restriction endonuclease assay to detect ZFN-induced mutations in Arabidopsis protoplasts. Mutations introduced by NHEJ are resistant to restriction enzyme digestion due to loss of restriction sites and result in uncleaved PCR products (indicated by arrows). Genomic DNA was cleaved with restriction enzymes before PCR amplification to enrich for ZFN-induced mutations. Lanes: 1, protoplast sample transformed with ADH1 ZFN after 24 h incubation; 2, protoplast sample transformed with ADH1 ZFN after 48 h of incubation; 3, control protoplast sample without transformation; 4, protoplast sample transformed with TT4 ZFN after 24 h of incubation; 5, protoplast sample transformed with TT4 ZFN after 48 h of incubation; 6, control protoplast sample without transformation; (C) Restriction endonuclease assay to detect ZFN-induced mutations in Arabidopsis seedlings after estrogen induction. DNA fragments lacking restriction sites (arrows) are detected in experimental samples with estrogen induction but not in control samples without estrogen. In this case, enrichment by prior restriction digestion of genomic DNA was not performed. Lanes: 1, ADH1 ZFN transgenic T₁ plants without estrogen induction; 2, ADH1 ZFN transgenic T₁ plants with estrogen induction; 3, TT4 ZFN transgenic T₁ plants without estrogen induction; 4, TT4 ZFN transgenic T₁ plants with estrogen induction.

Fig. 4.

Sequences of germinally transmitted mutations induced by ZFNs. For each target gene, the wild-type sequence is shown at the top with the ZFN recognition sites underlined. The recovered mutant alleles are shown below the wild-type sequence. Deletions are indicated by dashed lines, and insertions are indicated by lowercase letters. (A) Germinal mutations at ADH1. The numbers in superscript (i.e., 1, 2 and 3) identify mutations derived from individual T₁ plants. In many cases, a given plant produced multiple independent mutations. For example, plant 1 produced exclusively adh1 double knock-out (i.e., biallelic) mutant progeny, and the two distinct mutant alleles are indicated by the superscript 1. (B) Germinal mutations at TT4. The numbers in superscript identify T1 plants that contain multiple independent mutations at both alleles of TT4.