Schematics of the four classes of sequence-specific nucleases.

<p>(A) The meganuclease, I-SceI, is shown bound to its DNA target. The catalytic domain, which also determines DNA sequence specificity, is shown in red. (B) A ZFN dimer is illustrated bound to DNA. ZFN targets are bound by two zinc-finger DNA binding domains (dark blue) separated by a 5–7-bp spacer sequence. FokI cleavage occurs within the spacer. Each zinc finger typically recognizes 3 bp. (C) Depicted is a TALEN dimer bound to DNA. The DNA binding domains are in dark blue. The two TALEN target sites are typically separated by a 15–20-bp spacer sequence. Like ZFNs, the TAL effector repeat arrays are fused to FokI. Each TAL effector motif recognizes one base. (D) The CRISPR/Cas9 system recognizes DNA through base pairing between DNA sequences at the target site and a CRISPR-based guide RNA (gRNA). Cas9 has two nuclease domains (shown by red arrowheads) that each cleave one strand of double-stranded DNA.</p

Inheritance of targeted mutations and Cas9 in progeny of primary CRISPR/Cas events.

<p>Three primary events with cloned targeted mutations (lanes 1, 8 and 12; underlined) were used to generate genetic populations to assess inheritance of targeted mutations. The diploid event (lane 1; X46-3) was crossed to an inbred diploid line, M6 (lane 18) as the female parent while tetraploid events (lanes 8, 12; D46-44, D46-9) were selfed. Six progeny from the X46-3 population (lanes 2–7) and three progeny from the D46-44 (lanes 9–11) and D46-9 (lanes 13–15) populations were assessed for <b>A</b>) targeted mutations using a restriction digestion assay (top gel) and inheritance of Cas9 (bottom gel) and used for <b>B</b>) cloning targeted mutations using previously described methods (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144591#pone.0144591.g002" target="_blank">Fig 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144591#pone.0144591.s005" target="_blank">S5 Fig</a>). The PCR assay used for detecting Cas9 (<b>A</b>; bottom gel) produced a 1144 bp amplicon with each lane corresponding to the top gel and is further described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144591#pone.0144591.s006" target="_blank">S6 Fig</a> Wild-type Désirée and M6 were used as negative controls (lanes 16 and 18, respectively) and a 1:1 template mixture with wild-type and mutated DNA was used as a positive control (lane 17). The lengths of deletions (-) or insertions (+) of the targeted mutations in progeny (<b>B</b>) are in parenthesis to the left of each cloned mutation and the number of reads generated in the primary event (F₀) or individual progeny (F₁) are in brackets on the right. All targeted mutations were aligned to wild-type sequence and cloned from <i>StALS1</i> unless indicated on the right. PAM sequences are in gray.</p

Generation of targeted mutations in callus tissues of potato using CRISPR/Cas reagents.

<p><b>A</b>. Target sites of single-guide RNA within potato <i>StALS1</i> and -<i>2</i> genes. A single nucleotide polymorphism (lowercase) exists in the gRNA746 target site of <i>StALS2</i> but not gRNA751. <i>Alo</i>I and <i>Bsl</i>I restriction enzyme sites exist in sgRNA target sites of both genes (underlined). Arrows indicate primers used for enrichment PCR and restriction enzyme digestion assays. PAM sequences are in gray. <b>B</b>. Modified enrichment PCR assay using potato callus tissue transformed with gRNA746 and gRNA751 CRISPR/Cas reagents. Total genomic DNA was subjected to PCR amplification of the <i>StALS</i> target site (bottom image; 448 bp), digested overnight with <i>Alo</i>I (lanes 1, 3, 5, 7, 9, 11) or <i>Bsl</i>I (lanes 2, 4, 6, 8, 10, 12), and reamplified (top image; 448 bp) to generate an enriched amplicon. Enriched band intensities were normalized by dividing the quantified band intensity of the enriched band by the primary PCR amplicon (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144591#pone.0144591.s008" target="_blank">S1 Table</a>). Positive (+), negative (-) and non-detectable (ND) enriched bands have normalized intensities equal or over 0.5, less than 0.5 and equal or more than 0.05, or less then 0.05, respectively. Diploid (X; lanes 1–6) and tetraploid (D; lanes 7–12) genotypes were tested using both sgRNAs in the conventional 35S (M; lanes 1, 2, 7, 8) and geminivirus LSL (L; lanes 3, 4, 9, 10) T-DNA backbones. Wild-type (wt; lanes 5, 6, 11, 12) genomic DNA was used as non-transformed controls.</p

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

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

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 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

Mutation size and frequency for mRNA and DNA delivery of SSNs.

<p>The bar graph illustrates the unique insertion profiles between samples transformed with mRNA and DNA reagents.</p

TALEN activity in <i>N</i>. <i>benthamiana</i> protoplasts.

<p>A) Schematic of <i>ALS2</i>, indicating the TALEN target site (black triangle). The target site is 306 bp downstream of the stop codon. B) Bar graph depicting the frequency (%) of NHEJ-induced mutations created by the ALS2T1 TALEN with the four different UTRs as well as the no-UTR control. Data for both mRNA and DNA constructs are presented. The asterisk denotes a sample that is significantly different from the no-UTR control (p = 0.0065). Error bars denote standard error. C) Representative NHEJ-induced mutations. In the wild-type (WT) sequence, the TALEN binding site is indicated as underlined, bold text. Representative mutations have the number of deleted bases indicated at the right.</p

Schematic of mRNA expression constructs.

<p>A) The organization of a generic mRNA expression plasmid is shown with the T7 promoter, nuclease expression cassette and a poly-A sequence. B) Cassettes are illustrated that were used to express mRNA of the desired coding sequence (CDS). The sizes of the UTRs are given in bp. Note that the CDS is not drawn to scale.</p