Targeted insertions achieved by co-injection of single-stranded oligonucleotides (ssODNs) and the RGN system.

<p>The sgRNAs targeting <i>fh</i> and <i>gsk3b</i> have been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068708#pone.0068708-Hwang1" target="_blank">[11]</a>. For each gene, the wild-type sequence is shown at the top with the target site highlighted in yellow and the PAM sequence highlighted as red underlined text. For some cases the target sites are highlighted in green if the target sequences are in the reverse complement strand. The ssODNs containing 3–4 nucleotide (nt) insertions are shown beneath the wild-type sequences. The targeted insertions are highlighted as blue underlined capital letters. The target gene sequences identified in the injected embryos are shown beneath the ssODN sequences. Some of them contain only the precise intended changes (labeled as “precise” in parentheses on the right), while others contain additional indel mutations (deletions are shown as red dashes highlighted in grey and insertions as lower case letters highlighted in blue). The number of times each mutant sequence was isolated is shown in brackets.</p

Engineered RGNs induces heritable gene disruption.

<p>(A) Schematic illustration of the RGN system. Engineered sgRNA:Cas9 system is depicted here based on the target sequence of the <i>fh</i> gene. sgRNA interacts with the complementary strand of the DNA target site harboring a 3′ protospacer adjacent motif (PAM) sequence (NGG) (yellow and red underlined text, respectively). sgRNA also interacts with Cas9 endonuclease (blue shape), resulting in DNA double-strand breaks (DSBs) at the target site. The reverse complement of the target site is highlighted as green text and the reverse complement of the PAM site is shown as red underlined text. The potential cleavage sites of Cas9 are indicated by arrowheads. This graphic representation is modified from a previous publication <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068708#pone.0068708-Hwang1" target="_blank">[11]</a>. (B) Mutation frequencies in the germline induced by engineered RGNs. Fish that have been injected with sgRNA and Cas9 mRNA at the 1-cell stage were screened for founders. The concentrations of the sgRNA and the Cas9 mRNA are as indicated. The somatic mutation rates induced by these combinations have been reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068708#pone.0068708-Hwang1" target="_blank">[11]</a>. The percentages of the injected embryos that developed normally at 1 day post-fertilization are shown. The sequences of the indel mutations identified in the germline are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068708#pone-0068708-g002" target="_blank">Figure 2</a>.</p

Single-nucleotide substitution achieved by co-injection of single-stranded oligonucleotides (ssODNs) and the RGN system.

<p>ssODNs carrying 1-nucleotide (nt) sequence substitutions (fh_MscI.S, fh_AgeI.S and fh_mPAM.S) were co-injected with Cas9 mRNA and the sgRNA targeting the <i>fh</i> gene. The wild-type <i>fh</i> sequence is shown at the top with the target site highlighted in yellow and the PAM sequence highlighted as red underlined text. The intended modifications are highlighted as blue underlined capital letters. The target gene sequences identified in the injected embryos are shown beneath the ssODN sequences. Some of them contain only the precise intended changes (labeled as “precise” in parentheses on the right), while others contain additional indel mutations (deletions are shown as red dashes highlighted in grey and insertions as lower case letters highlighted in blue). One of the identified sequence has a 1-bp point mutation (highlighted in bold and by an underline) in addition to the intended sequence. The number of times each mutant sequence was isolated is shown in brackets.</p

Somatic mutation frequencies induced by RGNs.

<p>(A) GGN18 and GGN20 sgRNAs and their genomic target sequences. Sequences of the variable regions of the sgRNAs are shown here. These sgRNAs contain 1–2 nt mismatches to their genomic target sequences at the 5′ end. sgRNAs bind to the reverse complement strand of the DNA that possess the genomic target sequences (see illustration in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068708#pone-0068708-g001" target="_blank">Figure 1A</a>). Matching genomic and sgRNA sequences are marked in red, while the mismatches are marked in blue. PAM is underlined. (B) The indel mutation frequencies were assess using the T7EI assay.</p

Non-homologous end joining repair of ZFN-generated double-strand breaks within the <i>ROSA26</i> locus.

<p>(A) Schematic of ZFN 90/91 and 204/205 target sites within <i>ROSA26</i> intron 1. ZFN pairs 90/91 and 204/205 target sites 75 bp and 403 bp upstream of the <i>Xba</i>I site (white arrows), which is routinely used in <i>ROSA26</i> targeting, respectively. ZFNs 204/205 target a partial <i>Fsp</i>I recognition sequence. RF and RR, <i>ROSA26</i> forward and reverse primers used for NHEJ analysis generating a 474 bp fragment (black arrows). (B) Screening for NHEJ repair at the ZFN204/205 cleavage site. Genomic DNA extracted from fetuses or pups developing from ZFN-injected zygotes was amplified with primers RF and RR and subjected to <i>Fsp</i>I restriction digest. Most error-prone NHEJ repair events eliminate the <i>Fsp</i>I recognition sequence (underlined in C) resulting in an indigestible band at 474 bp. In the majority of founders such as Z20 both modified and wt alleles were detected, however only mutated alleles were present in founder ZGFP112. (C) Cloning and sequencing of undigested PCR products reveals mutations around the ZFN204/205 cleavage site. Founder ZGFP112 carried an identical Δ23 deletion in both <i>ROSA26</i> alleles. ZFN 204/205 recognition sites highlighted in bold and the spacer region in grey color.</p

ZFN 204/205 promote <i>ROSA26</i> targeting by homologous recombination in mouse zygotes.

<p>(A) HR targeting strategy for the insertion of the targeting vector gtR26_EGFP carrying EGFP driven by a CAG promoter into the <i>ROSA26</i> locus. (B) Southern blot analyses of <i>Eco</i>RI digested genomic DNA from a GFP-fluorescent animal showing site-specific integration into the <i>ROSA26</i> locus. Both 5′ and 3′ probes detect only one expected fragment in the DNA of wild-type (wt) animal. Additional fragments detected in the DNA of targeted animal (ti) are consistent with the integration of the CAG-EGFP cassette into one of the <i>ROSA26</i> alleles. (C) Germline transmission of the <i>ROSA26-CAG-EGFP</i> allele was confirmed by junction PCR in two F1 mice, one of which is depicted in (D). Primers RF, GF, and RR2 generate a 2.5 kb fragment from <i>ROSA26</i> wt alleles, while an additional 3.2 kb fragment is amplified from a gtR26_EGFP targeted allele.</p

Compilation of zygote microinjection experiments.

<p>EGFP: enhanced green fluorescent protein, NHEJ: non-homologous end-joining, TV: targeting vector, HR: homologous recombination, sc: supercoiled, tdT: tdTomato, SA: splice-acceptor, conc: concentration; E: 15dpc embryos,</p>*<p>pRosa26.8 described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041796#pone.0041796-Meyer1" target="_blank">[28]</a>.</p

Targeted genome editing in RS-SCID fibroblasts.

<p>(<b>A</b>) Schematic of genome editing strategy. Homology-directed repair (HDR) between the <i>prkdc</i> locus and the donor DNA is promoted by ZFN cleavage in intron 84 (BS, binding site). The HDR donor consists of flanking homology arms (dashed lines), splice acceptor (SA), cDNA encoding <i>prkdc</i> exons 85 and 86, polyadenylation signal (pA), neomycin resistance cassette (<i>NeoR</i>). The SCID underlying mutation in exon 85 (mut*), and primer binding sites for PCR analysis (5’-junction J5-F/J5-R; 3’-junction J3-F/J3-R; allelic discrimination AD-F/AD-R; mRNA expression RT-F/RT-R) are indicated. (<b>B</b>) Genome editing. After transfection of SCID fibroblasts with various ratios of donor DNA to ZFN expression plasmids, successful gene targeting in polyclonal samples was detected by an inside-out PCR amplification of the genome–donor 5´-junction (J5-F/J5-R). (<b>C</b>) Expression of corrected <i>prkdc</i> mRNA. After transfection of SCID fibroblasts, successful splicing from exon 83 to cDNA was detected with an inside-out RT-PCR strategy using primers RT-F/RT-R.</p

<i>In vitro</i> differentiation of iPSCs to proT-cells and T-cells.

<p>(A) Schematic of <i>in vitro</i> T-cell differentiation from iPSCs. Differentiation of iPSCs starts with formation of embryoid bodies that are dissociated to give rise to hematopoietic stem and progenitor cells (HPC). DL-1 mediated Notch signaling coaxes HPC development towards early proT-cells (DN2), which undergo DNA-PK dependent V(D)J recombination. After passing through DN3 and DN4 stages, preT-cells mature into double-positive (DP) T-cells that express the beta chain of the T-cell receptor (TCRß). Dashed lines indicate to what stage iPSC clones are expected to differentiate. (B) Assessment of T-cell differentiation. <i>In vitro</i> T-cell differentiation was analyzed by flow cytometry after two weeks of co-cultivation on OP9-DL1. Gating (indicated on top of each column) was applied in the following order: FSC/SSC and CD45⁺/DAPI^—to assess CD4/CD8 expression; CD8^–/CD4^—to gate for DN1-DN4 stage cells; CD8^–/CD4^– (DN) or CD8⁺/CD4⁺ (DP) to assess TCRß expression. Numbers indicate percentage of cells in each quadrant. HPC, lineage-negative bone marrow cells; iPS.WTX, wild-type iPSC; iPS.S6X, SCID iPSC clone; iPS.T25, gene targeted SCID iPSC clone.</p

Targeted genome editing in SCID-derived iPSCs.

<p>(<b>A</b>) Verification of gene targeting. Inside-out PCR strategies (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005239#pgen.1005239.g001" target="_blank">Fig 1A</a>) were used to verify correct 5´ (J5) and 3´-junctions (J3) of the integrated donor. Allelic discrimination (AD) PCR was used to assess mono- vs. bi-allelic integration. Targeted allele runs at 2.99 kb. Sizes of all expected PCR amplicons are indicated on the right. iPS.WT, wild-type iPSC; iPS.S6, SCID iPSC clone; iPS.T8, iPS.T25, iPS.T44, iPS.T45 and iPS.T60, targeted SCID iPSC clones. (<b>B</b>) Expression of corrected <i>prkdc</i> mRNA. Successful splicing from exon 83 to cDNA encompassing exons 84/85 was detected by an inside-out RT-PCR strategy using primers RT-F/RT-R.</p