Journal: Scientific reports

Article Title: Induction of site-specific chromosomal translocations in embryonic stem cells by CRISPR/Cas9.

doi: 10.1038/srep21918

Figure Lengend Snippet: Figure 1. Strategy for generating cellular and mouse models of chromosomal translocation via the ESC- and CRISPR/Cas9-based technologies. (a) Strategy for generating mESC models, or mESC-derived cellular models, and mouse models carrying a chromosomal translocation. (b) Strategy for generating site-specific chromosomal translocations in mESCs using the CRISPR/Cas9 system. Cdx2 and Gsk3α sgRNAs will guide Cas9 (blue) onto the indicated target sites located in mouse chromosome 5 (red) and chromosome 7 (green), respectively. DSBs will then be induced in these two sites. By activating NHEJ, DSBs can be repaired and the chromosomal translocation T (5:7) may occur in the designated location, thus generating two translocated chromosomes. To show the precise location and the relative length of the chromosomes, the chromosome graphs from the University of California Santa Cruz (UCSC) Genome Browser were used. Primer chr-short-p1 was designed to anneal to chromosome 7 at the site upstream of the predicted DSB point. Primer chr-short-p2 was designed to anneal downstream of the chromosome 5 DSB point. The size of PCR product is expected to be approximately 930 bp if the translocation occurs. Similarly, primers chr-long-p1 and chr-long-p2 were designed to detect T (5:7) chromosome-long, and the size of the PCR product is approximately 300 bp.

Article Snippet: Human codon optimized Cas9 gene was cloned from hCas9 plasmid (Addgene plasmid 41815) and inserted into the revised piggyBac expression vector with hygromycin resistance.

Techniques: Translocation Assay, CRISPR, Derivative Assay

Journal: Scientific reports

Article Title: Induction of site-specific chromosomal translocations in embryonic stem cells by CRISPR/Cas9.

doi: 10.1038/srep21918

Figure Lengend Snippet: Figure 2. Translocation between chromosome 5 and chromosome 7 mediated by the CRISPR/Cas9. (a) PCR analysis with chr-short-p1 and chr-short-p2 primers showing the presence of a ~930 bp PCR product in E14-Cas9 mESCs infected with Cdx2 and Gsk3α -sgRNAs. (b) Sequence of the PCR product (in one pMD18-T clone) of the predicted T (5:7) chromosome-short, and one cytosine nucleotide was deleted at the junction point. (c) PCR analysis with chr-long-p1 and chr-long-p2 primers showing the presence of a ~300 bp PCR product in E14-Cas9 mESCs infected with Cdx2 and Gsk3α sgRNAs. (d) Sequencing of the PCR product (in one pMD18-T clone) of the predicted T (5:7) chromosome-long indicates the addition of five nucleotides at the junction point. (e) Fluorescent images of the metaphase chromosomes of mESCs labelled with chromosome 5 (red) and 7 (green) specific probes. Insets zoomed in the two translocated chromosomes. Scale bars represent 10 μ m.

Article Snippet: Human codon optimized Cas9 gene was cloned from hCas9 plasmid (Addgene plasmid 41815) and inserted into the revised piggyBac expression vector with hygromycin resistance.

Techniques: Translocation Assay, CRISPR, Infection, Sequencing

Journal: Nature

Article Title: Saturation Editing of Genomic Regions by Multiplex Homology-Directed Repair

doi: 10.1038/nature13695

Figure Lengend Snippet: (a) Experimental schematic. Cultured cells were co-transfected with a single Cas9-sgRNA construct (CRISPR) and a complex homology-directed repair (HDR) library containing an edited exon that harbors a random hexamer (blue, green, orange) and a fixed selective PCR site (red). CRISPR-induced cutting stimulated homologous recombination with the HDR library, inserting mutant exons into the genomes of many cells. At five days post-transfection, cells were harvested for gDNA and RNA. After reverse transcription, selective PCR was performed followed by sequencing of gDNA and cDNA derived amplicons. Hexamer enrichment scores were calculated by dividing cDNA counts normalized by gDNA counts. (b) Correlation of enrichment scores between biological replicates for hexamers observed in each experiment with positions of previously identified exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs) and stop codons indicated. (c) Rank-ordered plot of enrichment scores with positions of ESEs, ESSs, and stop codons indicated.

Article Snippet: A bicistronic Cas9-sgRNA vector designed to cleave within BRCA1 exon 18 (“pCas9-sgBRCA1x18”) was cloned according to a published protocol by ligating annealed oligonucleotides into a human codon-optimized S. pyogenous Cas9-sgRNA vector from the lab of Feng Zhang (pX330-U6-Chimeric_BB-CBh-hSpCas9; Addgene plasmid #42230).

Techniques: Cell Culture, Transfection, Construct, CRISPR, Random Hexamer Labeling, Homologous Recombination, Mutagenesis, Sequencing, Derivative Assay

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Schematic of an ssDNA molecule (purple) tethered between two optically trapped micrometer‐sized polystyrene beads (grey) with hRAD51 complexes (green) bound to the ssDNA molecule. By controlling the position of the beads, the extension of the DNA molecule can be controlled while the tension in the molecule is monitored. At the same time, the proteins can be directly visualized with single‐fluorophore resolution using wide‐field fluorescence microscopy. The experiments are generally performed using a microfluidic flow system with four laminar channels. A typical experiment is comprised of the following steps: (1) capture of two beads; (2) tethering of a single dsDNA molecule between these beads; (3) probing the mechanical properties of the tethered dsDNA molecule, to ensure that it is a single molecule with the expected mechanical properties; (4) the tension on the dsDNA molecule is increased to generate an ssDNA molecule by force‐induced melting; (5) the ssDNA is incubated in the protein channel; and (6) the hRAD51‐ssDNA complex is brought into the buffer for imaging. Typical fluorescence intensity snapshots of hRAD51‐ssDNA complexes (buffer composition: 20 mM Tris pH 7.5, 100 mM KCl, 1 mM MgCl 2 , 1 mM ATP, 10 mM DTT) at indicated time intervals. Scale bars: 2 μm. Fluorescence kymograph of the same hRAD51‐ssDNA complex as in (C). Scale bars: 2 μm (horizontal) and 5 min (vertical). Integrated fluorescence intensity along the DNA of the same hRAD51‐ssDNA complex as in (C and D) over time (red dataset), showing an exponential decay at a rate of (33 ± 1) 10 −4 s −1 , obtained from an exponential fit to the data (blue curve). After correcting for photobleaching, this gives, for this particular example, a hRAD51 disassembly rate of (17 ± 1) 10 −4 s −1 . Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Fluorescence, Microscopy, Incubation, Imaging

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Fluorescence images and kymographs of hRAD51 disassembling from ssDNA at indicated ssDNA tensions (buffer composition: 20 mM Tris pH 7.5, 100 mM KCl, 1 mM MgCl 2 , 1 mM ATP, 10 mM DTT). Images are typical examples of 16 (at 5 pN), 9 (at 20 pN) and 4 (at 50 pN) identical experiments. Scale bars: 2 μm (horizontal) and 5 min (vertical). Integrated fluorescence intensity along the DNA of the same complexes as in (A) over time and corresponding an exponential fit. Fits are normalized using the amplitude and offset of the exponential fit. Coloured edges in (A) show colour of corresponding force curve. Average disassembly rate as a function of tension. Error bars originate from the exponential fits on the individual datasets (number of datasets as in A). Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Fluorescence

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Fluorescence images and kymographs of hRAD51 disassembling from ssDNA for filaments assembled in 20 mM Tris pH 7.5, 40 mM KCl, 10 mM Mg(OAc) 2 , 2 mM ATP and 10 mM DTT and disassembled in 20 mM Tris pH 7.5, 100 mM KCl, 1 mM MgCl 2 , 1 mM ATP and 10 mM DTT (right panel) or filaments assembled in 20 mM Tris pH 7.5, 40 mM KCl, 10 mM Mg(OAc) 2 , 2 mM ADP and 10 mM DTT and disassembled in 20 mM Tris pH 7.5, 100 mM KCl, 1 mM MgCl 2 , 1 mM ADP and 10 mM DTT (right panel). Images are typical examples of 16 (for the ATP condition) and 11 (for the ADP condition). Scale bars: 2 μm (horizontal) and 5 s (vertical). Normalized integrated fluorescence intensity of the images shown in (A) over time. Exponential fits to these traces give disassembly rates of (6.7 ± 0.3) 10 −4 s −1 (black dataset; in the presence of ATP) and (17 ± 4) 10 −4 s −1 (red dataset; in the presence of ADP). Coloured edges in (A) show colour of corresponding force curve. Disassembly rate is correlated with the initial coverage of the DNA molecule. Red closed circles: measured in ATP at 5 pN; red open circles: measured in ATP at 20 pN; red closed triangles: measured in ATP at 50 pN; red open triangles: measured in ATP at 75 pN; black closed circles; measured in ADP in 100 mM KCl and 1 mM MgCl 2 ; black open circles; measured in ADP in 100 mM KCl and 10 mM MgCl 2 ; black triangles: measured in 1 mM MgCl 2 . Blue line: linear fit with a slope of (−21 ± 3) 10 −10 s −1 /AU (Pearson's correlation coefficient of the fit: −0.84) that was used to correct observed disassembly rates for differences in initial coverage. Average disassembly rates in ATP or ADP conditions after correcting for differences in initial coverage. Since the error bars of the black and red datasets overlap, there is no significant difference between the disassembly rate in the presence of ATP or ADP. All coverages were scaled to 400,000 using a linear approximation based on the fit in (C). Data information: Error bars: SEM, based on 11 molecules (black dataset) and 16 molecules (red dataset). Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Fluorescence

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Fluorescence images and kymographs of hRAD51 disassembling from ssDNA of filaments assembled in 20 mM Tris pH 7.5, 40 mM KCl, 10 mM Mg(OAc) 2 , 2 mM ADP and 10 mM DTT and disassembled in 20 mM Tris pH 7.5, 1 mM MgCl 2 , 1 mM ADP and 10 mM DTT (left panel); filaments assembled in 20 mM Tris pH 7.5, 40 mM KCl, 10 mM Mg(OAc) 2 , 2 mM ADP and 10 mM DTT and disassembled in 20 mM Tris pH 7.5, 100 mM KCl, 1 mM MgCl 2 , 1 mM ADP and 10 mM DTT (middle panel); and filaments assembled in 20 mM Tris pH 7.5, 40 mM KCl, 10 mM Mg(OAc) 2 , 2 mM ADP and 10 mM DTT and disassembled in 20 mM Tris pH 7.5, 100 mM KCl, 10 mM MgCl 2 , 1 mM ADP and 10 mM DTT (right panel). Images are typical examples of 5, 11 and 3 identical experiments, respectively. Scale bars: 2 μm (horizontal) and 5 s (vertical). Normalized integrated fluorescence intensity of the images shown in (A) over time. Exponential fits to these traces give disassembly rates of (8 ± 1) 10 −4 s −1 (black dataset), (6.7 ± 0.3) 10 −4 s −1 (red dataset) and (6.7 ± 0.2) 10 −4 s −1 (blue dataset). Coloured edges in (A) show colour of corresponding force curve. Curves are typical examples of 5, 11 and 3 identical experiments, respectively. Average disassembly rates at indicated ionic strengths do not vary significantly. Error bars: SEM, based on 5, 11 and 3 identical experiments, respectively. Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Fluorescence

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: A Force‐extension/force‐relaxation cycles of a hRAD51‐coated ssDNA molecule in ATP and Ca 2+ (buffer: 20 mM Tris pH 7.5, 2 mM CaCl 2 , 2 mM ATP, 1 mM DTT). Time difference between cycles as indicated. Note that extension and relaxation curves of ssDNA (grey dataset) completely overlap. Arrows: direction in which the curves were recorded. Grey: ssDNA. Inset: fluorescence images with edges coloured as force curves. Representative example of two identical experiments. Scale bars: 2 μm. B Normalized integrated fluorescence intensity of the same construct over time. The amount of DNA‐bound hRAD51 remains constant; that is, photobleaching is negligible during these experiments. Representative example of two identical experiments. C The hysteresis area, defined as the area between extension and relaxation curve, remains roughly constant over time under these conditions. Representative example of two identical experiments. D Successive extension–relaxation cycles measured in the presence of Ca 2+ , Mg 2+ and ATP, showing reversible curves up to around 10 pN (first blue region), as all ATP‐bound NPFs are in ATP‐compact state. At higher forces, ATP‐bound NPFs transition to the ATP‐extended state and the curves show hysteresis (red region). Above 50 pN, all NPFs are in the ATP‐extended conformation (second blue region). Representative example of two identical experiments. E The relaxation curve is reversible. After converting all ATP‐bound NPFs into the ATP‐extended conformation and relaxing the molecule to 24 μm (black), the DNA is extended (red) before complete relaxation (blue). Red and blue curves overlap, showing reversibility of the relaxation curve. Representative example of two identical experiments. F Example of an eWLC fit (green) to a relaxation and extension curve (black) and corresponding contour length–extension curve (red) measured in the presence of ATP and Ca 2+ . Representative examples out of 38 experiments. eWLC fit parameters: persistence length 2.501 ± 0.009 nm, stretch modulus 1,599 ± 8 pN and contour length 29.01 ± 0.01 μm (error bars originate from individual eWLC fits). Contour length–extension curves were calculated under the assumption that upon conversion from ATP‐compact to ATP‐extended conformation, persistence length and stretch modulus remain constant. G–I Same as (A–C) but in presence of ADP, Ca 2+ and Mg 2+ (buffer: 20 mM Tris pH 7.5, 10 mM Mg(OAc) 2 , 2 mM CaCl 2 , 2 mM ADP, 1 mM DTT). Here, there is no ATP hydrolysis but hRAD51 can disassemble from the ssDNA. We see no hysteresis (G) and (I), while the integrated fluorescence intensity decreases (H) exponentially, yielding a disassembly rate of (4.2 ± 0.3) 10 −4 s −1 . Representative example of two identical experiments. Scale bars: 2 μm. Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Fluorescence, Construct

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Force‐extension and force‐relaxation curve measured in a buffer containing ATP and Mg 2+ . Under these conditions, ATP hydrolysis and NPF disassembly can occur. We observe a slight hysteresis between extension and relaxation curves. However, the disassembly rate under these conditions is relatively high, such that the assumption that the amount of hRAD51 bound remains constant during one extension–relaxation cycle is no longer valid. Therefore, under these conditions, no quantitative analysis (such as shown for other conditions in Fig ) of the hysteresis and the structural transitions can be performed. Scale bar: 2 μm. Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques:

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: A–C Same as Fig A–C but recorded in the presence of ATP, Ca 2+ and Mg 2+ (buffer composition: 20 mM Tris pH 7.5, 10 mM Mg(OAc) 2 , 2 mM CaCl 2 , 2 mM ATP, 1 mM DTT). Under these conditions, ATP hydrolysis can occur, and thus, hRAD51 can disassemble from the ssDNA. Therefore, the difference between the extension and relaxation curves (A), the total fluorescence intensity (B) and hysteresis area (C) decrease over time. In (A), the blue curve is indistinguishable from the grey curve of bare ssDNA. The disassembly rate can be determined either by an exponential fit to the fluorescence data (B), yielding, after correcting for photobleaching, a rate of (3.5 ± 0.3) 10 −4 s −1 , or by an exponential fit to the hysteresis data (C), yielding a rate of (4 ± 1) 10 −4 s −1 . Data shown is a representative example of six identical experiments. D–F Same experiments as in (A–C) in the presence of Ca 2+ and Mg 2+ (20 mM Tris pH 7.5, 2 mM CaCl 2 , 10 mM Mg(OAc) 2 , 1 mM DTT). NPFs were formed in the presence of ATP, but there was no ATP or ADP in the observation channel. Under these conditions, ATP hydrolysis can occur, and thus, hRAD51 can disassemble from the ssDNA, but reloading of ATP to the NPF after ATP hydrolysis and ADP release is impossible. The differences between the extension and relaxation curves (D), the total fluorescence intensity (E) and hysteresis area (F) decrease over time. In (D), blue curve is indistinguishable from grey curve of bare ssDNA. The disassembly rate can be determined by an exponential fit to the data in (E), giving a rate of (4 ± 1) 10 −4 s −1 , or by an exponential fit to the data in (F), giving a rate of (4 ± 1) 10 −4 s −1 . Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Fluorescence

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: A, B Side (A) and top (B) views of the asymmetric unit content of the crystal, which consists of 14 ATP‐bound copies of hRAD51 arranged as two heptameric right‐handed filaments. The two heptameric oligomers form continuous helical filaments running through the crystal. The hRAD51 molecules are drawn as ribbons, colour‐coded in blue or green in the two heptamers, with cylinders marking the position of each alpha helix. The ATP molecule is drawn as spacefill model. The positions of the N‐terminal and ATPase domains of RAD51 are indicated in (A). The RAD51 chains in the two heptameric assemblies are labelled A to G and H to N in (B).

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques:

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Structural superposition of the 12 dimers present in the asymmetric unit of the hRAD51‐ATP filament. Each hRAD51 dimer is coloured in light blue or yellow, according to the interface type between its two protomers. The protein chains of the dimers are drawn as a C α backbone, with one ATP moiety shown as stick model. The illustration underneath the superposition reports the distribution of interface types between protomers in the two hRAD51‐ATP heptamers, with RAD51 chains labelled A to G and H to N, as in Fig B. Each interface is marked by a coloured bar, colour‐coded blue or yellow as in the structural superimposition. The relative displacement in the position of hRAD51 residues between the two dimer types is illustrated by drawing the hRAD51 C α backbone as tube of varying radius, in direct proportion to the rmsd value for each amino acid (a larger radius corresponds to a higher rmsd). The structure of the reference hRAD51 structure used in the superposition is drawn as a spacefill model. Two filament structures of different pitch and rise are obtained by modelling a filament based exclusively on one or the other dimer type found in the crystal structure of the hRAD51‐ATP filament (see also and ). Seven hRAD51 protomers corresponding approximately to one helical turn are shown in both cases, drawn as molecular surfaces in light blue.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques:

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Based on the experiments involving stretching cycles such as shown in Figs A–I, , and G and H, we propose a cartoon model with the following conformational states and possible transitions. Because of the large hysteresis in the data shown in Fig A–C, we propose that there are two ATP‐bound states: an ATP‐compact and an ATP‐extended state with a free‐energy difference of (4 ± 1) k B T. Because there is no significant hysteresis in the experiments shown in Fig G–I, we propose that there is only one ADP‐bound state, from which the hRAD51 NPF can disassemble: the ADP state. For switching between ATP‐bound and ADP‐bound states, additional energy is required. This is generated by the hydrolysis of one ATP molecule (providing ~25 k B T). Black: ssDNA, blue: ATP‐compact hRAD51 monomer, green: ATP‐extended hRAD51 monomer, purple: hRAD51 monomer in ADP state, red: ATP, yellow: ADP.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Generated