Targeted DNA excision in Arabidopsis by a re-engineered homing endonuclease

Abstract

Background: A systematic method for plant genome manipulation is a major aim of plant biotechnology. One approach to achieving this involves producing a double-strand DNA break at a genomic target site followed by the introduction or removal of DNA sequences by cellular DNA repair. Hence, a site-specific endonuclease capable of targeting double-strand breaks to unique locations in the plant genome is needed.

Results: We engineered and tested a synthetic homing endonuclease, PB1, derived from the I-CreI endonuclease of Chlamydomonas reinhardtii, which was re-designed to recognize and cleave a newly specified DNA sequence. We demonstrate that an activity-optimized version of the PB1 endonuclease, under the control of a heat-inducible promoter, is capable of targeting DNA breaks to an introduced PB1 recognition site in the genome of Arabidopsis thaliana. We further demonstrate that this engineered endonuclease can very efficiently excise unwanted transgenic DNA, such as an herbicide resistance marker, from the genome when the marker gene is flanked by PB1 recognition sites. Interestingly, under certain conditions the repair of the DNA junctions resulted in a conservative pairing of recognition half sites to remove the intervening DNA and reconstitute a single functional recognition site.

Conclusion: These results establish parameters needed to use engineered homing endonucleases for the modification of endogenous loci in plant genomes.

Figure 1

DNA-protein interactions of endonucleases and their in vitro cleavage of distinct DNA substrates. (A) Diagram of wild-type I-CreI homodimer in complex with its natural DNA recognition sequence. One I-CreI monomer is shown in white, the other (I-CreI’) in grey. DNA sequence is indicated, with four base-pair center sequence shown in bold. Direct hydrogen bonds between I-CreI and DNA are shown as black lines. Sites of phosphodiester bond cleavage and the resulting 4 bp 3^′ overhangs are indicated by a line. A likely unfavorable electrostatic interaction between E80 and a backbone phosphate is indicated by a small arrow. (B) Predicted interactions between rationally designed PB1 endonuclease and the RS_GTAC DNA site. The two monomers (PB1 and PB1^′) and DNA interactions are as indicated in (A), except amino acids that deviate from I-CreI and I-CreI’ hydrogen bonds (or a hydrophobic interaction, C33) and are predicted to contribute to altered DNA-cleavage specificity are indicated with dashed lines. PB1+ endonuclease contains a mutation (E80 to Q80) predicted to eliminate the unfavorable interaction mentioned in (A). (C) Cleavage of DNA by native and rationally designed endonucleases. I-CreI, PBI, and PB1+ endonucleases were incubated with three distinct linearized DNA substrates (sequence indicated above its respective set of digests). Sequence differences between I-CreI (wild-type) and the two PB1 recognition sites highlighted in grey. DNA for PB1_TAGA (center) and PB1_GTAC (right) differ from each other by the 4 bp center sequence (subscript). Digests were conducted with 0, 0.007, 0.015, 0.031, 0.062, 0.125, 0.25, 0.5, 1, 2 μM endonuclease.

Figure 2

In planta cleavage of PB1 recognition sites by engineered endonucleases. (A) T-DNA structure before and after induction of the endonuclease. Endonuclease cleavage excises the central fragment 5^′-TTCTGCAG-3^′, eliminating the indicated PstI site. RB, right border; HSP, Hsp18.2 promoter; Endo, PB1 or PB1+, endonuclease; T, Nopaline Synthase terminator; RS, endonuclease recognition site (RS_TAGA or RS_GTAC); Kan, kanamycin resistance marker; LB, left border. Horizontal arrows indicate approximate locations of PCR primers used for diagnostic evidence of in planta endonuclease cleavage. (B) Table of experimental results. Seven different T-DNA constructs used in this study, with the general form diagramed in (A). Each T-DNA has three possible differences: presence (Yes) or absence (No) of a nuclear localization signal (NLS) on the endonuclease; the endonuclease with either the lower activity PB1 or higher activity PB1+ (containing Q80E mutation) PB1 recognition sites (RS) contain either a TAGA or GTAC central 4 bp sequence. Plants containing some constructs (JJS20, 23, and 26) had a low recovery rate after heat shock treatment, resulting in a lower number of plants screened. (C) Sample agarose gel data showing loss of the PstI restriction site from genomic DNA following heat-shock treatment of JJS24 plants. The agarose gel shows three JJS24 samples that demonstrated loss of the PstI site. Control (C) shows size of uncut PCR fragment. PCR fragments from samples before heat shock (–) are cut >90% into smaller bands (identified as “cut” on left). After heat shock (+), PCR fragments from the three samples are largely uncut by PstI, indicating a loss of the PstI site in planta.

Figure 3

Induction of PB1+ endonuclease removes BAR gene from Arabidopsis plants. (A) Schematic of the JJS30 T-DNA before and after induction of the PB1+ endonuclease. Two RS_GTAC sites flank the BAR gene, so that induction of the endonuclease excises the herbicide resistance gene from the genome. The heat-inducible promoter Hsp18.2 controls expression of PB1+. Arrows indicate location of PCR primers used to assay for BAR excision. (B) PCR analysis of JJS30 primary transformants before and after heat-shock, using primers shown in (A). Unmodified JJS30 T-DNA yields a PCR product approximately 1200 bp in length (BAR+), whereas JJS30 lacking the BAR gene is approximately 300 bp (BAR–). (C) DNA sequence of repair junctions from BAR– clones. The approximately 300 bp PCR products from (B) were cloned and sequenced to evaluate the DNA repair junctions. Forty-six clones were evaluated that represented ten plants yielding a significant amount of BAR minus (-) PCR product (excluding plants 2 and 11). Ten unique sequences were obtained and these are aligned with the “perfect re-ligation” product (sequence 1), in which the reconstituted PB1 recognition site is shaded and the location of phosphodiester bond cleavage/re-ligation is indicated by the arrowhead. Total number of independent clones that yielded each sequence is indicated, as well as the number of individual transformed plants that yielded those clones. Bases that are conserved between the two halves of the repair junction (microhomology) are underlined. Single and double base insertions at the repair junction are shown in lower case (sequence 8 and 4, respectively).