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: Cancer Discovery

Article Title: Transcriptional Silencing of ALDH2 Confers a Dependency on Fanconi Anemia Proteins in Acute Myeloid Leukemia

doi: 10.1158/2159-8290.cd-20-1542

Figure Lengend Snippet: Figure 3. Inactivation of FA genes in AML leads to p53-induced cell-cycle arrest and apoptosis. A, Representative flow cytometry analysis of BrdU incorporation and DNA content to infer cell status following lentiviral transduction of MOLM-13 cells with the indicated sgRNAs (day 6). B, Quantifica- tion of different cell-cycle stages, average of three biological replicates. Paired Student t test was applied to calculate P values. C, Representative flow cytometry analysis of DAPI (indicating permeable dead cells) and Annexin-V staining (a preapoptotic cell marker) following lentiviral transduction of MOLM-13 cells (day 6). D, Quantification of live and apoptotic cells, average of three biological replicates. Paired Student t test was applied to calculate P values. E, Gene set enrichment analysis of RNA-seq data obtained from MOLM-13 cells lentivirally transduced with the indicated sgRNAs (70). Normalized enrichment score (NES) and family-wise error rate (FWER) P value are shown. F, Western blot analysis performed on lysates obtained from MOLM-13 cells on day 6 following sgRNA transduction. G, Competition-based proliferation assays in MOLM-13 cells following sequential sgRNA transduc- tion. Negative sgRNA or TP53 sgRNAs were infected first, selected with neomycin, followed by transduction with the sgRNAs indicated at the bottom of the graph (linked with GFP). n = 3. All bar graphs represent the mean ± SEM. All sgRNA experiments were performed in Cas9-expressing cell lines.

Article Snippet: Plasmid Construction: sgRNA and shRNA Cloning For CRISPR screening, the optimized sgRNA lentiviral expression vector (LRG2.1T) and the lentiviral human codon–optimized Streptococcus pyogenes Cas9 vector (LentiV_Cas9_Puro, Addgene: 108100) were used.

Techniques: Flow Cytometry, BrdU Incorporation Assay, Transduction, Staining, Marker, RNA Sequencing, Western Blot, Infection, Expressing

Journal: PLoS ONE

Article Title: CRISPR/Cas9 as Tool for Functional Study of Genes Involved in Preimplantation Embryo Development

doi: 10.1371/journal.pone.0120501

Figure Lengend Snippet: Developmental competence and targeting efficiency of CRISPR/Cas9 mediated Porcine OCT4 locus. Cas9 mRNA and sgRNAs were injected in combinations of different concentrations, and cleavage and blastocyst formation rates of each groups are presented.

Article Snippet: A previously described plasmid (pCAG-T3-hCAS-pA)[ ], which contains human codon-optimized Cas9 plasmid cloned into a T3 promoter, was obtained from Addgene (Addgene #41815) and used to generate Cas9 mRNA.

Techniques: CRISPR, Injection, Sequencing

Journal: PLoS ONE

Article Title: CRISPR/Cas9 as Tool for Functional Study of Genes Involved in Preimplantation Embryo Development

doi: 10.1371/journal.pone.0120501

Figure Lengend Snippet: (A) Design of sgRNAs for targeting exon 2 or exon 5. Guide sequences correspond to sgRNAs are marked as underlined and the protospacer adjacent motif(PAM) sites for each guide sequences are marked. (B) Sequencing of PCR amplification product confirmed the introduction of indel in exon 2 or exon 5. Locations of guide sequences are marked as blue and bases deleted in Cas9/sgRNAs injected embryos are marked in red box. (C-D) Various deletion/insertions induced by Cas9/sgRNA injections in exon 2 (C) or exon 5(D). Positions of PAM sites are marked as underlined and deleted base was marked as red. Note that insertion can be induced in some case(marked in blue). (E-F) Detections of OCT4 and CDX2 protein using immunostaining in targeted porcine embryos. Embryos were injected with 5–10pl of 100ng/μl of Cas9 mRNA mixed with 0, 10, 100ng/μl of sgRNA1 or sgRNA2, respectively. Location of nucleus was stained with Hoechst 33342 (blue). OCT4 (left panel) and CDX2(right panel) are presented as green. (G) mRNA expression levels of OCT4 , CDX2 , and NANOG measured by qRT-PCR. Expression levels are presented as relative expression levels to those in control embryo at 1 cell stages. Control: Cas9 mRNA injection only; CRISPR: Cas9 mRNA and sgRNA 2 injected. In each developmental stages, 20 embryos were collected for RNA extraction.

Article Snippet: A previously described plasmid (pCAG-T3-hCAS-pA)[ ], which contains human codon-optimized Cas9 plasmid cloned into a T3 promoter, was obtained from Addgene (Addgene #41815) and used to generate Cas9 mRNA.

Techniques: Sequencing, Amplification, Injection, Immunostaining, Staining, Expressing, Quantitative RT-PCR, CRISPR, RNA Extraction

Journal: PLoS ONE

Article Title: CRISPR/Cas9 as Tool for Functional Study of Genes Involved in Preimplantation Embryo Development

doi: 10.1371/journal.pone.0120501

Figure Lengend Snippet: Developmental competence and targeting efficiency of CRISPR/Cas9 mediated large-scale deletions in Porcine OCT4 locus.

Article Snippet: A previously described plasmid (pCAG-T3-hCAS-pA)[ ], which contains human codon-optimized Cas9 plasmid cloned into a T3 promoter, was obtained from Addgene (Addgene #41815) and used to generate Cas9 mRNA.

Techniques: CRISPR, Sequencing

Journal: PLoS ONE

Article Title: CRISPR/Cas9 as Tool for Functional Study of Genes Involved in Preimplantation Embryo Development

doi: 10.1371/journal.pone.0120501

Figure Lengend Snippet: (A) Location of sgRNA pair in OCT4 locus and scheme for resulting deletion (B) Sequencing of PCR amplification product confimed the deletion of 1.8kb region between exon 2 and exon 5 of OCT4 locus. Locations of guide sequences in exon 2 and exon 5 are marked as blue(exon 2) or red (exon 5) respectively, and resulting exon 2—exon 5 fusions are shown. (C, D) Deletion of OCT4 and CDX2 protein using immunostaining in targeted porcine embryos. Embryos were injected with 5–10pl of 100ng/μl of Cas9 mRNA mixed with 0, 10, 100ng/μl of sgRNA 1 and sgRNA 2, respectively. Location of nucleus was stained with Hoechst 33342 (blue). OCT4 (C) and CDX2 (D) are presented as green.

Article Snippet: A previously described plasmid (pCAG-T3-hCAS-pA)[ ], which contains human codon-optimized Cas9 plasmid cloned into a T3 promoter, was obtained from Addgene (Addgene #41815) and used to generate Cas9 mRNA.

Techniques: Sequencing, Amplification, Immunostaining, Injection, Staining

Journal: PLoS ONE

Article Title: CRISPR/Cas9 as Tool for Functional Study of Genes Involved in Preimplantation Embryo Development

doi: 10.1371/journal.pone.0120501

Figure Lengend Snippet: (A) Scheme for the HDR-mediated integration of eGFP into OCT4 locus. Donor vector for HDR was consisted with the left homology arm (1kb) spanning exon 3—exon 5, eGFP fused after exon 5 as in-frame fusion and the right homology arm (1kb). Guide sequence for sgRNA was designed near stop codon of OCT4 located at exon 5. Location of PAM are underlined and stop codon of OCT4 is marked. Note that eGFP was inserted between stop codon and last codon of OCT4 and PAM of sgRNA was located just after stop codon. Therefore donor vector cannot recognize and digested with Cas9/sgRNA. (B) Confirmation of insertion of eGFP locus in genomic DNA of porcine embryo. PCR amplification spanning exon 5 confirmed the presence of in-frame fusion of eGFP at the end of OCT4 coding region. (C) Expression of eGFP fused Oct4 in HDR mediated eGFP knockin porcine embryos. eGFP expression was detected byConfocal microscopy. Control (Cas9 injected) and Targeted (Cas9/sgRNA/Donor Plasmid) have been compared. Note that localization of eGFP signal in nucleus.

Article Snippet: A previously described plasmid (pCAG-T3-hCAS-pA)[ ], which contains human codon-optimized Cas9 plasmid cloned into a T3 promoter, was obtained from Addgene (Addgene #41815) and used to generate Cas9 mRNA.

Techniques: Plasmid Preparation, Sequencing, Amplification, Expressing, Knock-In, Microscopy, Injection

Journal: PLoS ONE

Article Title: CRISPR/Cas9 as Tool for Functional Study of Genes Involved in Preimplantation Embryo Development

doi: 10.1371/journal.pone.0120501

Figure Lengend Snippet: Developmental competence and eGFP knockin efficiency in Porcine OCT4 locus.

Article Snippet: A previously described plasmid (pCAG-T3-hCAS-pA)[ ], which contains human codon-optimized Cas9 plasmid cloned into a T3 promoter, was obtained from Addgene (Addgene #41815) and used to generate Cas9 mRNA.

Techniques: Knock-In, Plasmid Preparation

Journal: Genome Research

Article Title: Whole-genome analysis of noncoding genetic variations identifies multiscale regulatory element perturbations associated with Hirschsprung disease

doi: 10.1101/gr.264473.120

Figure Lengend Snippet: Functional impacts of a HSCR-associated SNP (rs2435357) and the deletion of a novel S-HSCR enhancer on RET expression. (A) ATAC-seq and ChIP-seq profiles of hPSC and hNC in intron 1 of RET show that rs2435357 is residing in a hNC-specific ATAC-seq peak. (B) Location of rs2435357 in the RET gene locus and in the sgRNA used for CRISPR-Cas9-mediated HDR for editing the C allele to the HSCR-associated risk allele T. The electrographs of Sanger sequencing show the successful introduction of the risk allele at rs2435357 in the UE-rs2435357 hPSC line. (C) Differentiation strategy to generate human neural crest (hNC) and neuronal progenitor (hNP). HU is encoded by the ELAVL4 gene. Immunostaining of SOX10 and TUJ1 in hNC and hNP of the control and the mutant (UE-rs2435357) lines. Scale bars: (hNC) 100 μm; (hNP) 200 μm. RT-qPCR analysis showing the comparable ELAVL4 expression level in hNP in the control (n = 5) and the mutant (UE-rs2435357) (n = 3) lines. t-test, (ns) not significant. (D) RT-qPCR analysis showing RET expression in the hPSC and hNP stages of the control (n = 5) and the mutant (UE-rs2435357) (n = 3). t-test, (ns) not significant. (E) Hi-C data from neural progenitor cells show that the enhancer in intron 1 of RASGEF1A (marked in yellow on the right) has physical interaction with the promoter of RET (marked in yellow on the left) at 10-kbp bin size. (F) ATAC-seq and ChIP-seq data from hPSC and hNC at the RASGEF1A intron 1 locus. (G) The design of sgRNAs used for the CRISPR-Cas9 system for deleting the DNA fragment in RASGEF1A intron 1. Genotyping reveals the specific deletion of RASGEF1A intron 1 in the UE-RASGEF1A-int1-KO hPSC line. (WT) Wild type, (KO) knockout. (H) Immunostaining of SOX10, TUJ1, and HU in hNC and hNP of the control and the mutant (RASGEF1A-int1-KO) lines, respectively. Scale bars: (hNC) 100 μm; (hNP) 200 μm. (I) RT-qPCR reveals the expression level of RET in the hPSC and hNP stages of the control (n = 4–5) and the mutant (RASGEF1A-int1-KO) (n = 6–7). t-test, (ns) not significant.

Article Snippet: Human codon-optimized high fidelity Cas9 nuclease construct ( Ran et al. 2013 ) was obtained from Addgene (48138).

Techniques: Functional Assay, Expressing, ChIP-sequencing, CRISPR, Sequencing, Immunostaining, Control, Mutagenesis, Quantitative RT-PCR, Hi-C, Knock-Out

Journal: Genome Research

Article Title: Whole-genome analysis of noncoding genetic variations identifies multiscale regulatory element perturbations associated with Hirschsprung disease

doi: 10.1101/gr.264473.120

Figure Lengend Snippet: Characterization of a novel S-HSCR-associated regulatory element in intron 10 of PIK3C2B. (A) Overview of ATAC-seq and ChIP-seq profiles showing the putative hNC-specific regulatory element in PIK3C2B intron 10. The red shaded region indicates the location of the regulatory element and the line shows the A > T variant (rs551359143) found exclusively in the S-HSCR cases that disrupts the NFIA binding motif. The motif is not drawn to the same scale as the genomic signal tracks, with magnified characters. (B) Design of sgRNAs used for the CRISPR-Cas9 system for deleting the regulatory element. Genotyping reveals the specific deletion of the 171-bp fragment in intron 10 of PIK3C2B in the PIK3C2B-int10-KO hPSC line. (WT) Wild type, (KO) knockout. (C) Immunostaining shows that both the control and mutant (PIK3C2B-int1-KO) lines have comparable capability to make hNCs and hNPs. Scale bars: (hNC) 100 μm; (hNP) 200 μm. (D) RT-qPCR shows the changes in the expression of PIK3CB in different cell stages in the control and mutant lines. t-test, (ns) not significant. n = 3–4 per group. (E) Design of the constructs used for the luciferase assay. The bar chart shows the relative luciferase activities when the cells were transfected with different sets of constructs as indicated. Three independent assays were performed, each in triplicate. One-way ANOVA. (F) Gel mobility shift assays were performed with biotin-labeled probes containing the PIK3C2B intron 10 regulatory element with or without the A > T conversion and the nuclear extract from NFIA-overexpressing cells, in the presence of unlabeled probes or anti-NFIA antibody (0.1 µg). (G) Significant contacts (FDR < 0.05) in the promoter capture Hi-C data from GM12878 cells at the PIK3C2B locus. The putative regulatory element in intron 10 of PIK3C2B is marked in yellow. Contacts between the regulatory element and the TSSs of SOX13, PPP1R15B, and PIK3C2B are shown in purple curves, while contacts between the regulatory element and other promoters are shown in gray curves. Contacts that extend too far are trimmed. (H) RT-qPCR analysis shows the changes in the expression of PPP1R15B and SOX13 in the control and the mutant lines at different cell stages. t-test, (ns) not significant. n = 3–4 per group.

Article Snippet: Human codon-optimized high fidelity Cas9 nuclease construct ( Ran et al. 2013 ) was obtained from Addgene (48138).

Techniques: ChIP-sequencing, Variant Assay, Binding Assay, CRISPR, Knock-Out, Immunostaining, Control, Mutagenesis, Quantitative RT-PCR, Expressing, Construct, Luciferase, Transfection, Mobility Shift, Labeling, Hi-C