Targeted mutagenesis using zinc-finger nucleases in Arabidopsis - PubMed (original) (raw)

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

Alan Lloyd et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Targeted mutagenesis is an essential tool of reverse genetics that could be used experimentally to investigate basic plant biology or modify crop plants for improvement of important agricultural traits. Although targeted mutagenesis is routine in several model organisms including yeast and mouse, efficient and widely usable methods to generate targeted modifications in plant genes are not currently available. In this study we investigated the efficacy of a targeted-mutagenesis approach based on zinc-finger nucleases (ZFNs). In this procedure, ZFNs are used to generate double-strand breaks at specific genomic sites, and subsequent repair produces mutations at the break site. To determine whether ZFNs can cleave and induce mutations at specific sites within higher plant genomes, we introduced a construct carrying both a ZFN gene, driven by a heat-shock promoter, and its target into the Arabidopsis genome. Induction of ZFN expression by heat shock during seedling development resulted in mutations at the ZFN recognition sequence at frequencies as high as 0.2 mutations per target. Of 106 ZFN-induced mutations characterized, 83 (78%) were simple deletions of 1-52 bp (median of 4 bp), 14 (13%) were simple insertions of 1-4 bp, and 9 (8%) were deletions accompanied by insertions. In 10% of induced individuals, mutants were present in the subsequent generation, thus demonstrating efficient transmission of the ZFN-induced mutations. These data indicate that ZFNs can form the basis of a highly efficient method for targeted mutagenesis of plant genes.

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Figures

Fig. 1.

Fig. 1.

Targeted mutagenesis using ZFNs. (A) Strategy for induction of mutations using ZFNs. ZFNs bind to their recognition sequence (Z) and generate DSBs. Repair of the DSBs by NHEJ frequently produces mutations (*) at the break site. (B) The HS::QQR-QEQ construct, which consists of the HS::QQR gene and its recognition sequence, QEQ. The HS::QQR gene consists of the QQR coding sequence (QQR) fused to a heat-shock promoter (HS) and a 3′ terminator sequence (Ter). (Upper) The narrow box downstream of HS::QQR is the QEQ sequence. (Lower) The QEQ sequence, which contains binding sites for the QQR ZFN (labeled QBS) and an intervening _Eco_RI site. Arrowheads indicate the sites of QQR cleavage, which are within the _Eco_RI site.

Fig. 2.

Fig. 2.

The QQR ZFN generates mutations at its recognition sequence in Arabidopsis cells. (A) Depiction of the QEQ sequence relative to the primers (P1 and P2) used to amplify the region. Primers P1 and P2 refer to primers NOS5NH and M13(-20), respectively (see Materials and Methods). PCR amplification with primers P1 and P2 produces a 400-bp product. Digestion of this PCR product with _Eco_RI produces fragments of 320 and 80 bp. (B) Gel assay to detect QQR-induced mutations. PCR with primers P1 and P2 was carried out, and the 400-bp PCR products were subjected to gel electrophoresis either with (+ lanes) or without (- lanes) prior _Eco_RI digestion. DNA fragments lacking _Eco_RI sites (400-bp, upper band) were detected in experimental seedlings (heat induced) but not in control seedlings (not heat induced).

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