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: PeerJ

Article Title: Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

doi: 10.7717/peerj.8362

Figure Lengend Snippet: List of Edit Plasmids for organelle transformation. See ‘Materials and Methods’ for details.

Article Snippet: Codon-optimized Cas9 gene (Cas9m) was synthesized along with the 71 bp-long minimal promoter and the 119 bp-long terminator of the COX2 gene , and was flanked with Pst I and Hind III sites (GenScript).

Techniques: Transformation Assay, Plasmid Preparation

Journal: PeerJ

Article Title: Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

doi: 10.7717/peerj.8362

Figure Lengend Snippet: See ‘Materials and Methods’ for details. (A) Overall structures of Edit Plasmids; (B) Structure of the Cas9 expression cassette; (C) Structure of the gRNA expression cassette; and (D) Structure of the donor DNA. Scales are provided for B–D.

Article Snippet: Codon-optimized Cas9 gene (Cas9m) was synthesized along with the 71 bp-long minimal promoter and the 119 bp-long terminator of the COX2 gene , and was flanked with Pst I and Hind III sites (GenScript).

Techniques: Expressing

Journal: PeerJ

Article Title: Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

doi: 10.7717/peerj.8362

Figure Lengend Snippet: (A) Schematic view of the psaA-E3 genomic region targeted by two gRNAs (vertical arrows) and the donor DNA composed of codon-optimized GFPc gene that is provided by the Edit Plasmid. The recognition sites of primers used for the amplification of the junction regions are indicated (C1–C4). (B) PCR amplification of the junction region of the replaced DNA. Pooled DNAs extracted from independent colonies was used as templates with primers C2 and C4. Total number of colonies for each Edit Plasmids was 20 for YP13, 17 for YP14, 10 for YP21, 10 for YP22, 16 for YP23 and 24. Template for Lane 14 was untransformed wild-type cells. C2/C4 amplicon was 852 bp long. M: 1 kb plus molecular weight marker (New England Biolabs, Ipswich, MA, USA). (C) De-convolution of junction-PCR positive pools of YP13 transformants. A total of 12 events of the positive pools from Lanes 2 and 3 of (B) were analyzed by two primer sets C2/C4 and C1/C3 to amplify the left and right junction regions, respectively. Events #2 and #9 carried replaced DNA. C1/C3 amplicon was 712 bp long. (D) The sequence obtained from PCR amplification of the replacement DNA locus in Chlamydomonas plastid DNA modified by the Edit Plasmid approach. Underlined sequences: wild-type chloroplast genomic sequence that are not present on the Edit Plasmid. Sequences in bold: homologous regions (HR1c and HR2c) present in the donor DNA on the Edit Plasmid. Sequences in bold underlined: modified gRNA target sites present in the donor DNA. Sequences with double underlines: Silent mutations at the 3′ side of guide RNA sites to preclude re-cleavage by Cas9/sgRNA. (D) The sequence obtained from PCR amplification of the replacement DNA locus in Chlamydomonas plastid DNA modified by the Edit Plasmid approach. Underlined sequences: wild-type chloroplast genomic sequence that are not present on the Edit Plasmid. Sequences in bold: homologous regions (HR1c and HR2c) present in the donor DNA on the Edit Plasmid. Sequences in bold underlined: modified gRNA target sites present in the donor DNA. Sequences with dotted underlines: silent mutations at the 3′ side of guide RNA sites to preclude re-cleavage by Cas9/sgRNA.

Article Snippet: Codon-optimized Cas9 gene (Cas9m) was synthesized along with the 71 bp-long minimal promoter and the 119 bp-long terminator of the COX2 gene , and was flanked with Pst I and Hind III sites (GenScript).

Techniques: Plasmid Preparation, Amplification, Molecular Weight, Marker, Sequencing, Modification

Journal: PeerJ

Article Title: Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

doi: 10.7717/peerj.8362

Figure Lengend Snippet: Summary of Cas9/gRNA induced integration of donor DNA into organelle genomes. Numbers in parentheses: Independent transgenic events tested in Chlamydomonas chloroplasts and biological replications tested in yeast mitochondria.

Article Snippet: Codon-optimized Cas9 gene (Cas9m) was synthesized along with the 71 bp-long minimal promoter and the 119 bp-long terminator of the COX2 gene , and was flanked with Pst I and Hind III sites (GenScript).

Techniques: Transgenic Assay, Plasmid Preparation

Journal: PeerJ

Article Title: Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

doi: 10.7717/peerj.8362

Figure Lengend Snippet: DNA Sequences (5′–3′) near the cleavage site in cloned amplicons lacking the Ava II site are aligned with the wild-type parent DNA shown on the top line. Deduced amino acid sequence is shown under each DNA sequence. The 20-nucleotide target sequence for the Cas9/sgRNA complex is indicated in blue. The PAM site (in green), the Ava II recognition site (in bold blue), and SNPs and the resulting amino acid changes (in red) are also labeled.

Article Snippet: Codon-optimized Cas9 gene (Cas9m) was synthesized along with the 71 bp-long minimal promoter and the 119 bp-long terminator of the COX2 gene , and was flanked with Pst I and Hind III sites (GenScript).

Techniques: Clone Assay, Sequencing, Labeling

Journal: PeerJ

Article Title: Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

doi: 10.7717/peerj.8362

Figure Lengend Snippet: Summary of DNA alterations at sgRNA2c site in Chlamydomonas chloroplasts. SNP frequency was measured at the Ava II site in chloroplast transformants with Edit Plasmids carrying different Cas9 promoters ( psaA , psbD or none), vector backbone, and with and without the guide RNA (sgRNA2c) or donor DNA. SNP frequency was deduced by the number of SNP detected per number of amplicon clones analyzed (see ‘Materials and Methods’).

Article Snippet: Codon-optimized Cas9 gene (Cas9m) was synthesized along with the 71 bp-long minimal promoter and the 119 bp-long terminator of the COX2 gene , and was flanked with Pst I and Hind III sites (GenScript).

Techniques: Plasmid Preparation, Amplification, Clone Assay, Construct, Negative Control

Journal: PeerJ

Article Title: Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

doi: 10.7717/peerj.8362

Figure Lengend Snippet: See “Materials and Methods” for details. (A) Overall structures of Edit Plasmids; (B) Structure of the Cas9 expression cassette; (C) Structure of the gRNA expression cassette; and (D) Structure of the donor DNA. Scales are provided for B–D.

Article Snippet: Codon-optimized Cas9 gene (Cas9m) was synthesized along with the 71 bp-long minimal promoter and the 119 bp-long terminator of the COX2 gene , and was flanked with Pst I and Hind III sites (GenScript).

Techniques: Expressing

Journal: PeerJ

Article Title: Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

doi: 10.7717/peerj.8362

Figure Lengend Snippet: (A) Schematic view of the COX1 genomic region targeted by two gRNAs (arrows) and the donor DNA with GFP gene that is provided by the Edit Plasmid. The recognition sites of primers used for the amplification of the junction regions are indicated (C, F, 11 and 12). (B) PCR analysis of the junction regions of the integrated donor DNA. Left: 5′ region amplified with C/12 primer set; right: 3′ region amplified with F/11 primer set. Lanes 1–5: Five control lines with HS6 Edit Plasmid without Cas9 activity; lanes 6–10; Five lines with HS8 Edit Plasmid with Cas9 activity. (C) Wild-type CUY563 strain; (M) 1 kb plus molecular weight marker (New England Biolabs, Ipswich, MA, USA). Arrows: the size expected from the deduced sequence with integrated donor DNA (C/12 amplicon: 870 bp; F/11 amplicon: 907 bp). DNA fragments separated in lanes 6 and 10 for the both amplicons were isolated for sequence confirmation (see text). (C) The sequence obtained from PCR amplification of the replacement DNA locus in transformed yeast mitochondrial DNA modified by the Edit Plasmid approach. Underlined sequences: wild-type mitochondrial genomic sequences that are not present on the Edit Plasmid. Sequences in bold: short homologous regions present in the donor DNA (HR1 and HR2) adjacent to gRNA target sites. Sequences with dotted underlining: modified gRNA target sites present in the donor DNA (altered nucleotides are shown in bold). The codon-optimized GFP coding region is presented in italics. Sequences presented in lower case correspond to primers C and F that were used for amplification of the replacement DNA locus. Homologous recombination leading to DNA replacement occurred without causing any sequence changes either in the replacement DNA nor in the surrounding wild-type mitochondrial DNA.

Article Snippet: Codon-optimized Cas9 gene (Cas9m) was synthesized along with the 71 bp-long minimal promoter and the 119 bp-long terminator of the COX2 gene , and was flanked with Pst I and Hind III sites (GenScript).

Techniques: Plasmid Preparation, Amplification, Activity Assay, Molecular Weight, Marker, Sequencing, Isolation, Transformation Assay, Modification, Genomic Sequencing, Homologous Recombination

Journal: bioRxiv

Article Title: Efficient mutagenesis of maize inbreds using biolistics, multiplex CRISPR/Cas9 editing, and Indel-Selective PCR

doi: 10.1101/2024.10.21.619474

Figure Lengend Snippet: (A) Selfed ears of ILP1 harvested 12-14 days after pollination, with excised immature embryo in inset. (B) Putative transgenic calli on selective medium . (C) CRISPR/Cas9-events on selective regeneration medium. (D, E) Putative transgenic shoots on selective regeneration medium after 2- 3 weeks post-transfer to a growth chamber with 16h/8h light/dark photoperiod. (F) Sequence confirmation of genome edits in LW1 . The top half of the gel shows amplification of the 5’ end of the gene spanning the LW1 -gRNA4 target site, bottom half shows amplicons spanning target sites for LW1 -gRNA1, LW1 -gRNA2 and LW1 -gRNA3 at 3’ end of gene. Left lane in each gel is the NEB 1kb+ ladder. (G, H, I) Sanger sequencing results of PCR products shown in panel F (guide RNA targets are depicted in orange, PAM sequences are depicted in bold black letters; red dashes represent deletions, bold blue underlined letters represent insertions, and base changes are depicted in black underlined letters).

Article Snippet: The specific plasmid components used were a monocot optimized Cas9 and Csy4 protein expressed under the maize Ubiquitin1 promoter and separated by a P2A self-cleaving peptide sequence (Addgene ID: 91036), a multiplexed gRNA cassette expressed with a Cestrum Yellow Leaf Curling Virus (CmYCLV) promoter and separated by Csy4 cleavage sites (Addgene ID: 91061), a blank homologous recombination module (Addgene ID: 91081) and a plasmid backbone containing the NPTII resistance gene driven by the 2X 35S promoter (Addgene ID: 91203).

Techniques: Transgenic Assay, CRISPR, Sequencing, Amplification

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