Growth curves of CRISPR progeny.

<p>Eight heterozygous IgH KO6B females and eight control females were weighed weekly for 8 weeks.</p

Cre recombination of CRISPR-targeted loxP site.

<p>A. Diagram of the targeted IgH locus before and after Cre recombination. A forward primer upstream of the CRISPR-targeted loxP site was used with two different reverse primers downstream of the loxP site in the JH-KO cassette. In the non-recombined allele, the forward and reverse primers are separated by about 28kb on the chromosome. After Cre recombination, a single loxP site and the promoterless neo gene remain, and the primers are either 1.6 or 2kb apart, which amplifies readily. B. PCR of recombined cells. Cre +: gDNA template from 1783–9 cells transfected with Cre; Cre -, parental 1783–9 cells; JH-KO, gDNA from a heterozygous JH-KO bird; NTC, no template control.</p

Strategy for CRISPR targeting.

<p>A. Diagram of the chicken IgH locus present in PGC line 472–138 used for CRISPR targeting. The IgH locus contained a previously obtained knockout of the JH gene segment (JH-KO), between the D cluster and the constant regions (only Cμ is shown), which was replaced with a selectable marker cassette. gRNAs 1 through 4 were designed to target a region upstream of the single functional VH region (indicated with an arrow), and gRNA5 was designed to target the EGFP gene. B. PGCs of line 472–138 were transiently transfected with a construct containing Cas9 or Cas9/gRNA5, specific for EGFP. After 9 days in culture, the cells were analyzed by flow cytometry for loss of green fluorescence.</p

CRISPR-mediated targeting of IgH KO6B in PGCs.

<p>A. Detailed diagram of the IgH locus. The 122 bp sequence between the 5’ and 3’ homology regions in IgH KO6B, used to design the gRNAs, is shown at top. The locations of gRNAs 1–4 are indicated with blue lines above the sequence and the protospacer adjacent motifs (PAM) indicated with red lines. The repair vector IgH KO6B (below) contains 5’ and 3’ homology regions (HR) in yellow, a single loxP site (blue arrowhead), and a hygromycin selection cassette (orange). The locations of the primer binding sites for the 5’ and 3’ targeting assays are shown as black arrows. The downstream selectable markers in the JH-KO consist of floxed EGFP (green box) and puro gene (blue box), and a promoterless neo gene in opposite orientation (pink box). The loxP sites are blue arrowheads. B. The 5’ targeting assay performed on independent, non-clonal cell populations obtained from co-transfection of the four different gRNAs into 472–138 cells along with Cas9 and IgH KO6B. For each gRNA transfection, 3 hygromycin-resistant populations were analyzed. The positive control (+) was a DT40 cell line that contained a knockout of the functional V region [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154303#pone.0154303.ref022" target="_blank">22</a>] and the negative control (-) was the parental IgH KO6B plasmid. C. The 5’ and 3’ targeting assays performed on 9 independent clones obtained with gRNA2 (there were 12 clones, but clones 4, 7 and 12 grew more slowly and were not tested at this time). Variation in band intensity is likely to be from variation in the template gDNA amount, since the number of cells harvested was not normalized. The negative control (-) was genomic DNA from a JH-KO transgenic bird, and the positive control (+) was a pool of cells (G2) from the gRNA2 experiment in B. NT, no template control. D. The same 5’ and 3’ targeting assays performed on EGFP+ birds obtained from breeding cell line 1783–10 chimeras to wild type.</p

Germline transmission rates of chimeras made with CRISPR-modified PGCs.

<p>Germline transmission rates of chimeras made with CRISPR-modified PGCs.</p

Single-channel images related to Fig 8E and 8G.

(A) Fluorescence microscopic images of indicated strains expressing mNeonGreen-Pex15 (green), mCherry-PTS (magenta), and Sec63-CFP (blue). (B) Fluorescence microscopic images indicated strains expressing mNeonGreen-Pex15 (green), mCherry-PTS (magenta), and Tim50-CFP (blue). (C) Fluorescence microscopic images of control and indicated mutant cells expressing mNeonGreen-Pex15 (green) and mCherry-PTS (magenta). Scale bar represents 5 μm. (TIF)</p

Movement of peroxisomes in indicated strains.

Frames were captured with the following excitation times and gain settings, control, 75 ms, gain 23; Δpex30, 80 ms, gain 79; Δget2, 100 ms, gain 63; Δmdm10; 100 ms, gain 48. (MP4)</p

Genetic interactions of Mdm10.

(A) Indicated mutants expressing Pex3-GFP were analyzed by fluorescence microscopy (left). Quantifications of peroxisome number (right). (B) Indicated mutants expressing mCherry-PTS were analyzed by fluorescence microscopy (left). Quantifications of peroxisome number (right). Scale bars represent 5 μm. Quantifications are based on n = 3 experiments. Each color represents 1 experiment. Error bars represent standard error of the mean. P-values were calculated with a one-way ANOVA combined with a Tukey test. Underlying data for quantifications can be found in S1 Data. (TIF)</p

Quantification of Pxp2 at ERMES foci.

Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER–mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.</div

Overexpression of Pex15 derivatives induces contacts.

(A) Fluorescence microscopic images of cells co-expressing RFP-HDEL (magenta) PA-GFP-Pex15g (green) under control of the MET25 promoter grown in the presence of 200 μm methionine. (B) Fluorescence microscopic images of indicated strains expressing mNeonGreen-Pex15 (green) under control of the TEF1 promoter, mCherry-PTS (red), and Tim50-CFP (blue). (C) Induction of a chimeric RFP-Pex15-TATom22 (red) via galactose addition in strains expressing Ant1-YFP (green) and Tim50-CFP (blue). Scale bar represents 5 μm. (TIF)</p