Journal: Cell Death Discovery

Article Title: Human-specific gene CT47 blocks PRMT5 degradation to lead to meiosis arrest

doi: 10.1038/s41420-022-01139-6

Figure Lengend Snippet: A The procedures for generating CT47 mutations in hESCs. A plasmid system for sgRNA transcription and Cas9 expression. PGL3-U6-2 sgRNA-EF1a-Puro for sgRNA transcription and PSt1374-N-NLS-Cas9 for Cas9 expression. B FACS ploidy analysis of the haploid spermatogenic cell percentage of H1 and CT47 −/− SSCs in SSC culture with 200 pM testosterones. hESC and mouse sperm were used as control groups. C The results shown are the mean ± SD from two independent technical replicates in the indicated groups. Student’s t test: * p < 0.05, ** p < 0.01, N.S., no significant difference. D CT47 protein levels in human testicular biopsy samples from five nonobstructive azoospermia (NOA 1–5) and obstructive azoospermia (OA 1–5) patients. E Quantification of the western blot in D . The results shown are the mean ± SD, n = 5 in each of the two groups. Student’s t test: * p < 0.05. F CT47 expression levels from GSE145467. OA, obstructive azoospermia. NOA, Nonobstructive azoospermia. The results shown are the mean ± SD. Student’s t test: n = 10 per group, ** p < 0.01. G Schematic model showing that the dynamics of CT47 translocation and interaction with PRMT5 synchronizes the meiotic process of spermatocytes with testosterone during puberty.

Article Snippet: In brief, the Cas9 expression plasmid pST1374-Cas9-N-NLS-Flag linker (Addgene 44758, Cambridge, MA, USA) has been optimized for nuclear import by two nuclear localization sequences and contains cytomegalovirus (CMV) promoter for expression in human cells.

Techniques: Plasmid Preparation, Expressing, Control, Western Blot, Translocation Assay

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

Journal: International Journal of Molecular Sciences

Article Title: Gene Editing Correction of a Urea Cycle Defect in Organoid Stem Cell Derived Hepatocyte-like Cells

doi: 10.3390/ijms22031217

Figure Lengend Snippet: CRISPR/Cas9-mediated gene targeting of patient’s OTCD iPSC. ( a ) Genome engineering strategies. Single guide RNA (gRNA1, gRNA2) ( left panel ) and dual gRNA (combination of gRNA3a with gRNA3b, and gRNA4a with gRNA4b) ( right panel ) approaches were used to achieve the best editing efficiency. Single guides were delivered along with wild-type Cas9, while dual guides with nickase Cas9. Position of mutation respective to gRNAs is marked in the figure. ( b ) Assessment of cleavage efficiency. Editing efficiency was estimated based on intensities of wild-type and cleaved bands following restriction enzyme digestion. Negative: untransfected cells. CRISPR-transfected: cells transfected with Cas9 and gRNA. ( c ) Restriction enzyme assay—clone screening. A total number of 54 iPSC clones were serially isolated, expanded and screened. Screening was conducted with restriction enzyme digestion assay which would digest only successfully corrected DNA sequence of targeted region. One iPSC clone was edited on both alleles having only digested bands, similar to the healthy control (positive control). A representative unedited clone is also shown. ( d ) Sequencing of genomic DNA of unedited and edited clones. The genomic region of interest containing the disease-causing variant was Sanger-sequenced in unedited and edited iPSC clones. Three base pairs were identified as differences between the parental and the engineered cells (indicated with arrows), as expected. Two of those are silent mutations which were deliberately introduced to increase the correction efficiency, and one is the mutation intended to be edited to correct OTC deficiency. ( e ) Investigation of OTC transcript. Amplification of OTC transcript spanning from exon 1 to exon 5 was performed in OTC-proficient (OTCP) primary and OTCD primary hepatocytes, as well as in unedited and edited iPSC hepatocyte-like cells (iPSC-HLC). One wild-type band was presented in cells correctly expressing the gene (OTCP primary hepatocytes and edited iPSC-HLC), while two bands (wild-type and shorter band due to exon skipping) in genetically defected cells (OTCD primary hepatocytes and unedited iPSC-HLC).

Article Snippet: Two gRNAs were designed in order to be used with the wild-type Cas9 nuclease (pX458 vectors, Addgene, Watertown, MA, USA), and two pairs of gRNAs to be used with D10A nickase mutant version Cas9 (Cas9 nickase, Cas9n, vector pX461, Addgene, Watertown, MA, USA), as shown in a. Vectors expressed wild-type SpCas9 or D10A nickase SpCas9, tracrRNA and Green Fluorescent Protein (GFP), used for sorting positive-transfected cells.

Techniques: CRISPR, Mutagenesis, Transfection, Enzymatic Assay, Clone Assay, Isolation, Sequencing, Positive Control, Variant Assay, Amplification, Expressing