Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer

doi: 10.1007/s00018-014-1744-7

Figure Lengend Snippet: CRISPR/Cas9 mediates gene targeting in PFFs. a Schematic of sgRNAs targeting TYR, PARK2 and PINK1 loci. The target loci are located in the first exon following the start codon of these three genes. sgRNA targeting sites are highlighted in red. PAM are highlighted in blue and underlined. b Sanger sequencing of the targeting sites in mutant colonies used in SCNT. For each gene, the wile-type sequence is shown at the top with the target sites highlighted in red. The net change in length caused by each mutation is to the right of each sequence (+, insertion; ∆, deletion). Lowercase letters denote inserted base pairs. PAM is highlighted in blue. Indel insertion or/and deletion

Article Snippet: Construction of Cas9 and sgRNA vectors CMV promoter-driven Cas9 (41815) and Cas9-nickase (Cas9n, 41816) plasmids were purchased from Addgene.

Techniques: CRISPR, Sequencing, Mutagenesis

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer

doi: 10.1007/s00018-014-1744-7

Figure Lengend Snippet: CRISPR/Cas9-mediated gene targeting in PFFs

Article Snippet: Construction of Cas9 and sgRNA vectors CMV promoter-driven Cas9 (41815) and Cas9-nickase (Cas9n, 41816) plasmids were purchased from Addgene.

Techniques: CRISPR, Mutagenesis

Journal: The EMBO Journal

Article Title: PIF1 helicase promotes break‐induced replication in mammalian cells

doi: 10.15252/embj.2020104509

Figure Lengend Snippet: Schematic drawing of the EGFP‐BIR‐5085 reporter and the BIR repair product by SDSA (BIR‐SDSA) or end joining (BIR‐EJ) which results in EGFP expression after splicing. U2OS (EGFP‐BIR‐5085) reporter cell line was infected by lentiviruses encoding endonuclease I‐SceI or empty vector, followed by puromycin selection (2 µg/ml, 2 days) and assayed for the percentage of EGFP‐positive cells by FACS analysis 4 days post‐infection. BIR track length was determined in the single EGFP‐positive clones derived from U2OS (EGFP‐BIR‐5085) WT and PIF1 KO reporter cell lines after I‐SceI (left) or Cas9/sgRNA (right) cleavage by sequencing the PCR products of repair junctions using genomic DNA. Group means are shown and error bars represent ± SD. Dashed lines (3.8 and 0.9 kb) indicate the upper and lower limits of track length that can be scored by this reporter. U2OS (EGFP‐BIR‐5085) cells expressing shRNAs for RAD51, POLD3, or shRNA vector (Ctrl) were infected by lentiviruses encoding endonuclease I‐SceI. The percentage of EGFP‐positive cells was assayed by FACS analysis 4 days later (left). Expression of RAD51 and POLD3 is shown by Western blot analysis (right). U2OS (EGFP‐BIR‐5085) cells carrying Tet‐On Cas9/sgRNA‐5085 (Appendix Table ) and expressing indicated shRNA were treated by Nocodozale (0.3 μM) and 40 h later, Doxycycline (Dox, 5 μg/ml) was added. The percentage of EGFP‐positive cells was quantified by FACS analysis 48 h after induction (left). Cell cycles before and after Nocodazole treatment were analyzed by FACS following propidium iodide (PI) staining (middle). Expression of RAD51, POLD3, and RAD52 is shown by Western blot analysis (right). U2OS (EGFP‐BIR‐5085) cells expressing PIF1 shRNA or shRNA vector (Ctrl; left) or expressing PIF1‐WT or E307Q mutant with endogenous PIF1 depleted by shRNA (right) were infected by lentiviruses expressing I‐SceI. The percentage of EGFP‐positive cells was assayed by FACS analysis 4 days post‐infection. PIF1 expression level was determined by qPCR (Appendix Fig ), and the expression of PIF1‐WT or E307Q mutant is shown in Appendix Fig . U2OS (EGFP‐BIR‐5085) cells and two PIF1 knocked‐out (KO) clones derived from the U2OS (EGFP‐BIR‐5085) cell line and generated by CRISPR KO were assayed for the percentage of EGFP‐positive cells by FACS 4 days after I‐SceI lentiviral infection. Schematic drawing of the EGFP‐STGC‐1731 reporter and the repair product generated by STGC is shown (top). iEGFP: internal EGFP. U2OS (EGFP‐STGC‐1731) cells expressing RAD51, POLD3, PIF1 shRNA, or shRNA vector (Ctrl) were assayed for EGFP‐positive repair events by FACS analysis 4 days post‐infection of I‐SceI lentiviruses (middle). Expression RAD51 and POLD3 is shown by Western blot analysis (bottom). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by two‐tailed non‐paired parameters were applied in Student's t ‐test. The P value is indicated as ** P < 0.01, **** P < 0.0001 and n.s. (not significant) P > 0.05. Source data are available online for this figure.

Article Snippet: To obtain Tet‐On Cas9 (or Cas9n)/sgRNA cell lines, U2OS EGFP‐STGC‐1731, EGFP‐BIR‐5085, or EGFP/STGC‐mCherry/LTGC‐5034 reporter cell lines were infected with lentiviruses expressing Cas9 (or Cas9 D10A) and sgRNA from the lentiguide‐puro vector (Addgene #52963).

Techniques: Expressing, Infection, Plasmid Preparation, Selection, Clone Assay, Derivative Assay, Sequencing, shRNA, Western Blot, Staining, Mutagenesis, Generated, CRISPR, Standard Deviation, Two Tailed Test

Journal: The EMBO Journal

Article Title: PIF1 helicase promotes break‐induced replication in mammalian cells

doi: 10.15252/embj.2020104509

Figure Lengend Snippet: U2OS WT or PIF1 ‐KO cells were treated with the indicated concentrations of HU (left) or APH (right) for 72 h, and the cell viability assay was performed. U2OS WT or PIF1 ‐KO cells were labeled with CldU for 30 min followed by incubation with 2 mM HU for 2 h and then IdU for another 30 min. Labeled cells were processed for DNA fiber analysis. Representative images of stalled or restarted forks and forks with new origin firing were shown (left). The percentage of restarted forks was quantified by analyzing of 110–130 fibers for each experiment (right). Experiments were repeated four times for each sample. Schematic drawing of the EGFP‐BIR‐5085 reporter and the repair steps leading to the repair product expressing EGFP after Cas9n/sgRNA‐5085 expression. U2OS (EGFP‐BIR‐5085) cell lines carrying Dox‐inducible Cas9/sgRNA‐5085 (Dox‐Cas9) or Cas9n/sgRNA‐5085 (Dox‐Cas9n) were incubated with or without Dox (5 µg/ml) and assayed by FACS analysis 2 days later (left). U2OS (EGFP‐BIR‐5085, Dox‐Cas9 or Dox‐Cas9n) cells expressing shRNAs RAD51, POLD3, and PIF1 or shRNA vector (Ctrl) were incubated with 5 µg/ml Dox, and FACS analysis was performed after 2 days (right). Track length of single EGFP‐positive clones derived from U2OS (EGFP‐BIR‐5085) cells after Cas9/sgRNA‐5085 ( n = 47) or Cas9n/sgRNA‐5085 ( n = 39) expression was analyzed by sequencing of the PCR products from genomic DNA covering the repair junctions. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by two‐tailed non‐paired parameters were applied in Student's t ‐test. The P value is indicated as ** P < 0.01, *** P < 0.001.

Article Snippet: To obtain Tet‐On Cas9 (or Cas9n)/sgRNA cell lines, U2OS EGFP‐STGC‐1731, EGFP‐BIR‐5085, or EGFP/STGC‐mCherry/LTGC‐5034 reporter cell lines were infected with lentiviruses expressing Cas9 (or Cas9 D10A) and sgRNA from the lentiguide‐puro vector (Addgene #52963).

Techniques: Viability Assay, Labeling, Incubation, Expressing, shRNA, Plasmid Preparation, Clone Assay, Derivative Assay, Sequencing, Standard Deviation, Two Tailed Test

Journal: The EMBO Journal

Article Title: PIF1 helicase promotes break‐induced replication in mammalian cells

doi: 10.15252/embj.2020104509

Figure Lengend Snippet: Proposed models for repair of DSBs generated at broken replication forks (see text for details). seDSB: single‐ended DSB; deDSB: double‐ended DSB. Pink arrows: endonucleases to remove Flex1 or other secondary structures at DSB ends or DNA tails at the fork junctions. Schematic drawing of the EGFP‐STGC‐1731 reporter and proposed pathways to repair DSBs generated by endonuclease cleavage (I‐SceI or Cas9, left) or converted from nicks (Cas9n, right). U2OS (EGFP‐STGC‐1731) cell lines carrying Dox‐inducible Cas9/sgRNA‐1731 (Dox‐Cas9) or Cas9n/sgRNA (Dox‐Cas9n) were incubated with or without Dox (5 µg/ml), and the percentage of EGFP‐positive cells was quantified by FACS analysis 2 days later (top). U2OS (EGFP‐STGC‐1731, Dox‐Cas9 or Dox‐Cas9n) cells expressing shRNAs for RAD51, POLD3, and PIF1 or shRNA control (Ctrl) were incubated with Dox (5 µg/ml), and the percentage of EGFP‐positive cells was quantified by FACS analysis after 2 days (bottom). Schematic drawing of the EGFP‐Flex1‐STGC‐1541 reporter and the proposed mechanism to repair DSBs at Flex1 generated upon fork breakage by SDSA which involves two DSB ends. U2OS (EGFP‐Flex1‐STGC‐1541) cells were treated with or without 2 mM HU for 24 h, and the percentage of EGFP‐positive cells by HU induction was quantified by FACS analysis 4 days after removal of HU (left). U2OS (EGFP‐Flex1‐STGC‐1541) cells expressing shRNAs for RAD51, POLD3, and PIF1 or shRNA vector (Ctrl) were treated with 2 mM HU for 24 h, and the percentage of EGFP‐positive cells was quantified by FACS analysis 4 days later (right). U2OS (EGFP‐Flex1‐STGC‐1541) cells were infected by retroviruses expressing RAS or empty vector, and the percentage of EGFP‐positive cells was assayed by FACS 5 days following infection (left). U2OS (EGFP‐Flex1‐STGC‐1541) cells expressing shRNAs for RAD51, POLD3, and PIF1 shRNAs or shRNA vector were infected by retroviruses expressing RAS, and the percentage of EGFP‐positive cells was assayed by FACS 5 days after infection (middle). Expression level of RAD51 and POLD3 is shown by Western blot analysis (right). Proposed models for activating BIR at replication‐dependent and replication‐independent DSBs (see text for details). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by two‐tailed non‐paired parameters were applied in Student's t ‐test. The P value is indicated as ** P < 0.01 and n.s. (not significant) P > 0.05. Source data are available online for this figure.

Article Snippet: To obtain Tet‐On Cas9 (or Cas9n)/sgRNA cell lines, U2OS EGFP‐STGC‐1731, EGFP‐BIR‐5085, or EGFP/STGC‐mCherry/LTGC‐5034 reporter cell lines were infected with lentiviruses expressing Cas9 (or Cas9 D10A) and sgRNA from the lentiguide‐puro vector (Addgene #52963).

Techniques: Generated, Incubation, Expressing, shRNA, Control, Plasmid Preparation, Infection, Western Blot, Standard Deviation, Two Tailed Test

Journal: The EMBO Journal

Article Title: PIF1 helicase promotes break‐induced replication in mammalian cells

doi: 10.15252/embj.2020104509

Figure Lengend Snippet: Schematic drawing of EGFP/STGC‐mCherry/LTGC‐5034 reporter and the repair products generated by the STGC or LTGC/BIR mechanisms. U2OS (EGFP/STGC‐mCherry/LTGC‐5034) with Dox‐inducible Cas9/sgRNA‐1731 (Cas9) or Cas9n/sgRNA‐1731 (Cas9n) or empty vector (Vec) were assayed for recombination by FACS analysis 2 days after adding Dox (5 µg/ml). Representative FACS data (top), and the ratios of mCherry to EGFP in the indicated cell lines after Dox induction were shown (bottom). Track length of single mCherry‐positive clones derived from U2OS (EGFP/STGC‐mCherry/LTGC‐5034) cells after Cas9/sgRNA‐1731 or Cas9n/sgRNA‐1731 cleavage was analyzed by sequencing PCR products from genomic DNA at repair junctions. The numbers of the total events analyzed after Cas9 and Cas9n cleavage are shown on the top with the numbers of events using BIR‐EJ indicated in brackets. Group means are shown. Error bars represent ± SD. Dashed lines (2.2 and 1.1 kb) indicate the upper and lower limits of track length that can be scored by this reporter. U2OS (EGFP/STGC‐mCherry/LTGC‐5034, Dox‐Cas9 [left] or Dox‐Cas9n [right]) cells expressing shRNAs for POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP‐ or mCherry‐positive cells after induction was quantified by FACS analysis 2 days later to determine the percentage of EGFP‐ or mCherry‐positive cells. U2OS (EGFP‐Flex1‐STGC‐1541) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were treated with 2 mM HU for 24 h. The percentage of EGFP‐positive cells after HU treatment was quantified by FACS analysis 3 days after HU removal (left). U2OS (EGFP‐STGC‐1731) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were infected by lentivirus expressing I‐SceI. The percentage of EGFP‐positive cells by I‐SceI induction was quantified by FACS analysis 4 days later (right). U2OS (EGFP/STGC‐mCherry/LTGC‐5034, Dox‐Cas9 [left] or Dox‐Cas9n [right]) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP‐ or mCherry‐positive cells after induction was quantified by FACS analysis 2 days later. FLAG‐PIF1 was stably expressed in U2OS (EGFP‐Flex1‐STGC‐1541) cells by lentiviral infection. Enrichment of FLAG‐PIF1 at Flex1 site or GAPDH site with or without HU (2 mM, 10 h) treatment was calculated by anti‐FLAG ChIP (left). When PCNA was depleted by shRNA using vector as a control (Ctrl), enrichment of FLAG‐PIF1 at Flex1 site was calculated by anti‐FLAG ChIP (right). ChIP value in cells without HU treatment is set up as 1 for normalization. U2OS (EGFP‐Flex1‐STGC‐1541) cells expressing FLAG‐PIF1 WT or mutants with endogenous PIF1 depleted by shRNA were treated with 2 mM HU for 24 h. The percentage of EGFP‐positive cells after HU treatment was quantified by FACS analysis 3 days after HU removal. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by two‐tailed non‐paired parameters were applied in Student's t ‐test. The P value is indicated as ** P < 0.01 and n.s. (not significant) P > 0.05.

Article Snippet: To obtain Tet‐On Cas9 (or Cas9n)/sgRNA cell lines, U2OS EGFP‐STGC‐1731, EGFP‐BIR‐5085, or EGFP/STGC‐mCherry/LTGC‐5034 reporter cell lines were infected with lentiviruses expressing Cas9 (or Cas9 D10A) and sgRNA from the lentiguide‐puro vector (Addgene #52963).

Techniques: Generated, Plasmid Preparation, Clone Assay, Derivative Assay, Sequencing, Expressing, shRNA, Incubation, Infection, Stable Transfection, Control, Standard Deviation, Two Tailed Test