AID interacts with the calcium and integrin binding protein 1.

<p><b>A</b>. myc-CIB1 co-IPs AID-EGFP but not EGFP only (left and right lanes, respectively). A myc-specific monoclonal antibody was used to precipitate complexes, and AID-GFP was detected with an α-GFP polyclonal antibody. <b>B</b>. AID-EGFP co-IPs myc-CIB1 in a DNase I- and RNase A-resistant manner. An α-GFP monoclonal antibody was used to precipitate AID-GFP, and myc-CIB1 was detected with an α-myc monoclonal antibody. <b>C</b>. AID-STZ pulls down endogenous CIB1 from HEK-293T cell extracts. IgG Sepharose was used to pull-down STZ complexes, and CIB1 was detected using an α-CIB1 polyclonal antibody. AID-STZ and GFP-STZ were detected with an α-strep antibody. A low level of non-specific background was observed in the vicinity of AID-STZ. For cell lysate (input) control blots, two panels are shown because GFP-STZ is expressed over 100-fold better than AID-STZ. A quantification of the input versus pull-down signal indicated that 1% of cellular CIB1 can be pulled-down with AID complexes when IgG sepharose beads are limiting.</p

CIB1 is dispensable for CSR in mice.

<p><b>A</b>. Relative levels of each antibody isotype in sera from CIB1^+/+ or CIB1^−/− mice as measured by ELISA. The CIB1^−/− data were normalized to the mean antibody levels in sera from wildtype (WT) littermates (arbitrarily set to 1 for comparison). Each X represents data from an independent animal and the horizontal bars and labels report the median values (n = 3 for CIB^+/+ and n = 6 for CIB^−/−). <b>B</b>. IgM to IgG1 CSR <i>ex vivo</i>. B-cells were isolated from the spleens of CIB1^+/+ or CIB1^−/− mice, cultured for 4 days in the presence of LPS and IL-4, and analyzed by α-IgG1-PE labeling and flow cytometry. Each X represents data from an independent animal and the horizontal bars and labels report the median values. <b>C</b>. Images of hematoxylin and eosin stained sections of spleen isolated from CIB1^+/+ and CIB1^−/− mice. Scale bars indicate 500 µm.</p

CIB1 over-expression in DT40.

<p><b>A</b>. An IGC fluctuation analysis showing the percentage of surface Ig-positive cells in subclone cultures over-expressing human (h) or chicken (c) CIB1. Each X represents data from an individual subclone and the labeled horizontal bars report the medians for each data set. <b>B</b>. CIB1 over-expression confirmed by immunoblotting. Loading was controlled by stripping and re-probing the blot with an α-tubulin antibody.</p

AID localization in CIB^−/− DT40 cells.

<p><b>A</b>. AID-EGFP localization in CIB1^+/+ DT40. <b>B</b>. AID-EGFP localization in CIB1^−/− DT40. Images were taken using a 40× objective and the scale bars indicate 10 µm.</p

CIB1 is dispensable for immunoglobulin gene conversion in DT40.

<p><b>A</b>. Schematic showing the constructs used to replace exons 5 and 6 of <i>CIB1</i> with the indicated drug resistance cassettes. The positions of the allele-specific PCR primers for genotyping and the XhoI sites and the position of the 3′ external probe used for Southern blot analysis are shown. <b>B</b>. An agarose gel image showing the allele-specific PCR products from CIB1+/+, +/−, and −/− cell lines. <b>C</b>. An agarose gel image of CIB1-specific RT-PCR products from CIB1+/+, +/−, and −/− cell lines. AID-specific reactions were used to demonstrate the integrity of each cDNA preparation. <b>D</b>. An IGC fluctuation analysis showing the percentage of surface Ig-positive cells in subclone cultures of the indicated genotype. Each X represents data from an individual subclone and the labeled horizontal bars report the medians for each data set.</p

TAL Effector Specificity for base 0 of the DNA Target Is Altered in a Complex, Effector- and Assay-Dependent Manner by Substitutions for the Tryptophan in Cryptic Repeat –1

<div><p>TAL effectors are re-targetable transcription factors used for tailored gene regulation and, as TAL effector-nuclease fusions (TALENs), for genome engineering. Their hallmark feature is a customizable central string of polymorphic amino acid repeats that interact one-to-one with individual DNA bases to specify the target. Sequences targeted by TAL effector repeats in nature are nearly all directly preceded by a thymine (T) that is required for maximal activity, and target sites for custom TAL effector constructs have typically been selected with this constraint. Multiple crystal structures suggest that this requirement for T at base 0 is encoded by a tryptophan residue (W232) in a cryptic repeat N-terminal to the central repeats that exhibits energetically favorable van der Waals contacts with the T. We generated variants based on TAL effector PthXo1 with all single amino acid substitutions for W232. In a transcriptional activation assay, many substitutions altered or relaxed the specificity for T and a few were as active as wild type. Some showed higher activity. However, when replicated in a different TAL effector, the effects of the substitutions differed. Further, the effects differed when tested in the context of a TALEN in a DNA cleavage assay, and in a TAL effector-DNA binding assay. Substitution of the N-terminal region of the PthXo1 construct with that of one of the TAL effector-like proteins of <i>Ralstonia solanacearum</i>, which have arginine in place of the tryptophan, resulted in specificity for guanine as the 5’ base but low activity, and several substitutions for the arginine, including tryptophan, destroyed activity altogether. Thus, the effects on specificity and activity generated by substitutions at the W232 (or equivalent) position are complex and context dependent. Generating TAL effector scaffolds with high activity that robustly accommodate sites without a T at position 0 may require larger scale re-engineering.</p> </div

Targeted Mutagenesis in Plant Cells through Transformation of Sequence-Specific Nuclease mRNA

<div><p>Plant genome engineering using sequence-specific nucleases (SSNs) promises to advance basic and applied plant research by enabling precise modification of endogenous genes. Whereas DNA is an effective means for delivering SSNs, DNA can integrate randomly into the plant genome, leading to unintentional gene inactivation. Further, prolonged expression of SSNs from DNA constructs can lead to the accumulation of off-target mutations. Here, we tested a new approach for SSN delivery to plant cells, namely transformation of messenger RNA (mRNA) encoding TAL effector nucleases (TALENs). mRNA delivery of a TALEN pair targeting the <i>Nicotiana benthamiana</i> ALS gene resulted in mutation frequencies of approximately 6% in comparison to DNA delivery, which resulted in mutation frequencies of 70.5%. mRNA delivery resulted in three-fold fewer insertions, and 76% were <10bp; in contrast, 88% of insertions generated through DNA delivery were >10bp. In an effort to increase mutation frequencies using mRNA, we fused several different 5’ and 3’ untranslated regions (UTRs) from <i>Arabidopsis thaliana</i> genes to the TALEN coding sequence. UTRs from an <i>A</i>. <i>thaliana</i> adenine nucleotide α hydrolases-like gene (At1G09740) enhanced mutation frequencies approximately two-fold, relative to a no-UTR control. These results indicate that mRNA can be used as a delivery vehicle for SSNs, and that manipulation of mRNA UTRs can influence efficiencies of genome editing.</p></div

Activity of TAL effectors with selected single amino acid substitutions for W232 on targets with A, C, G, or T at the 0^th position.

<p>A. Schematic of a TAL effector with the -1^st and 0^th repeats and the central repeat region (CRR) labeled. The amino acid sequence of the -1^st repeat is shown below; W232 is shown in bold. B. Effects of PthXo1 W232 substitution and target combinations (treatment) on activity. Shown at top are the PthXo1 RVD and EBE sequences. X marks the 0^th position. Activity was measured in an <i>Agrobacterium</i>-mediated transient expression assay in <i>Nicotiana benthamiana</i> leaves, using a GUS reporter gene cloned downstream of a minimal promoter (see Materials and Methods) containing a PthXo1 EBE with the 0^th position thymine (EBE_PthXo1-T), or variants with adenine, cytosine, or guanine as base 0 (EBE_PthXo1-A, EBE_PthXo1-C, and EBE_PthXo1-G, and respectively). Treatment effects were estimated using a mixed linear model to account for variation due to experiment and replicate effects (see Materials and Methods). Effects were computed relative to the negative control EBE_PthXo1-T with no TAL effector and normalized to the effect of wild-type PthXo1 (W232) on EBE_PthXo1-T. Bars indicate one standard deviation. C. Effects of TAL868 W232 substitution and target combinations on activity, as in B. Shown at top are the TAL868 RVD and EBE sequences. X marks the 0^th position. </p

YFP expression in N. benthamiana protoplasts.

<p>Representative images of protoplasts ~24 hours after transformation. The top row shows protoplasts transformed with mRNA transcripts containing UTRs from the four genes tested. The lower row shows images for the controls, namely cells transformed with a DNA construct expressing YFP from a 35S promoter, YFP mRNA without UTRs and water. White arrowheads point to YFP expressing protoplasts.</p

Activity of chimeric TAL effector RTL-PthXo1 with single amino acid substitutions for R298 on targets with A, C, G, or T at the 0^th position.

<p>A. Schematic of the chimeric TAL effector RTL-PthXo1 with the -1^st and 0^th repeats and the central repeat region (CRR) labeled, and showing the RSc1815 -1st repeat sequence. RTL-PthXo1 was created by replacing the N terminal region immediately upstream of the CRR of our PthXo1 construct with the N terminal region of RTL RSc1815 (shown in red). The arginine (R) in the RTL that aligns to W232 is shown in large bold font. Numbers indicate positions in the RTL sequence. B. Left, activity of RTL-PthXo1 with the native arginine at position 298 (R298) and of variants with selected amino acid substitutions at that position (as labeled), measured in an <i>Agrobacterium</i>-mediated transient expression assay in <i>Nicotiana benthamiana</i> leaves, using a GUS reporter gene cloned downstream of a minimal promoter (see Materials and Methods) containing the PthXo1 EBE with the 0^th position thymine (EBE_PthXo1-T), or variants with adenine, cytosine, or guanine as base 0 (EBE_PthXo1-A, EBE_PthXo1-C, and EBE_PthXo1-G, and respectively). Right, the same plot at a larger scale. Activity is normalized to the activity of PthXo1 on EBE_PthXo1-T, set to 1.0. Values are the mean of five replicates. Error bars represent s.d. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082120#pone.0082120.s015" target="_blank">Table S9</a> for p-values. Experiments were repeated at least twice with similar results.</p