PMC

Inversion of the At1g53430 gene cluster. (A) Schematic of the At1g53430-At1g53440 gene cluster inversion. Positions of PCR primers for confirmation of inversions are indicated by empty or filled triangles and arrows. (B) PCR confirmation of gene cluster inversions. Two independent T2 lines were used to detect inversions; wild-type (WT) plants were used as the negative control. (C) Detailed depiction of the inversion event and DNA sequence confirmation of the inversions.

Deletion of gene clusters by ZFNs. (A) Deletion of the At1g53430-At1g53440 cluster by the At1g53-ZFN. (B) Deletion of the At1g70450-At1g70460 cluster by the At1g70-ZFN. (C) Three types of deletions at the At4g16960-At4g16860 gene cluster generated by the At4g16-ZFN. The gene clusters and resulting deletions are depicted. Deletion events were confirmed by PCR, as shown in windows in the middle of each panel. The positions of PCR primers are indicated by arrows. PCR products were subsequently cloned and sequenced. The sequencing results shown in the lower panels confirmed perfect ligations after loss of the intervening DNA or ligations with mutations at the ZFN cleavage site.

Schematic of target genes and ZFN sites. (A) The At1g53-ZFN targets both At1g53430 and At1g53440 in the 14th exon of each gene. (B) The At1g70-ZFN targets At1g70450 in the 1st exon and At70460 in the 2nd exon. (C) The At4g16-ZFN targets three sites in the RPP4 R gene cluster: the 1st exon of At4g16960, the 5′ UTR of At4g16940, and the 1st exon of At4g16860. Illustration of ZFN pairs depicts the DNA recognition triplets for each zinc finger. The zinc finger binding sequences are underlined, and the distance between cleavage sites is shown.

ZFN activity at seven endogenous loci. (A) At1g70-ZFN activity at target sites in At1g70450 and At1g70460. PCR products were digested with AflII. (B) At4g16-ZFN activity at target sites in At4g16960, At4g16940, and At4g16860. PCR products were digested with BfaI. (C) At1g53-ZFN activity at target sites in At1g53430 and At1g53440 as measured by the T7 endonuclease assay. For both restriction digestion assays (A and B) and the T7 endonuclease assay (C), mutagenesis frequencies (shown at the bottom of the figures) were determined by measuring the signal intensity of each band using the Labworks analysis software. (D-J) ZFN-induced mutations at seven endogenous target sites with different mutation types indicated. The restriction enzyme sites used for activity measurement are marked in bold letters. The uncut PCR products (from A and B, and Figure S2) were cloned and sequenced to reveal ZFN-induced mutations at each target site.

Large chromosomal deletions by ZFNs. (A) Schematic of ZFN targets on the right arm of Arabidopsis chromosome 1. The distance between the ZFN sites is shown and the positions of primers used to confirm large deletions are indicated. (B) PCR confirmation of large chromosomal deletions. The F1 and R primers amplify the junction fragment of the deletion of 9.037 Mb; primers F2 and R amplify the junction fragment of the deletion of 9.027 Mb (upper panels). PCR amplification of a part of the ADH1 gene was used as a genomic DNA control (lower panel). F1 seedlings generated from the cross between At1g53-ZFN #7 line and ADH1ZFN #3 line were treated with estradiol, and the wild-type plants served as a negative control. (C) Sequenced clones indicative of large chromosomal deletions between At1g53430 and At1g77120. (D) Sequenced clones indicative of large chromosomal deletions between At1g53440 and At1g77120. The DNA sequences resulting from perfect ligation of DNA ends are shown in the first line of the text boxes; deletions with indels are shown below. ZFN binding sequences are underlined. MH-NHEJ, end-joining that appears to have been facilitated by microhomology.