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: Journal of biotechnology

Article Title: Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris.

doi: 10.1016/j.jbiotec.2016.03.027

Figure Lengend Snippet: Fig. 1: Critical features for CAS9/gRNA expression affecting the genome editing efficiency. Implementing CRISPR/Cas9 in a given organism is reliant on a set of interdependent components. These features include the DNA sequence of CAS9, the type and position of the NLS for the nuclear import of Cas9, different gRNA sequences and target loci, the promoter for the expression of the gRNAs, the introduction of processing elements for RNA maturation (ribozymes) as well as transcriptional termination. Elements on the plasmid are not drawn to scale. Cas9 (blue) shown on the right side includes the gRNA (red) and the target DNA for cleavage (yellow). The illustration of the Cas9/gRNA complex was taken from the RCSB PDB (see acknowledgements).

Article Snippet: The Homo sapiens codon optimized CAS9 (HsCAS9) and the Streptococcus pyogenes CAS9 (SpCAS9) were amplified from the template vectors p414-TEF1p-Cas9-CYC1t and pMJ806 (both obtained from Addgene, Cambridge, MA, USA) using primer pairs spCas9_fw/spCas9-SV40_rv and hsCas9_fw/ hsCas9-SV40_rv respectively.

Techniques: Expressing, CRISPR, Sequencing, Plasmid Preparation

Journal: Journal of biotechnology

Article Title: Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris.

doi: 10.1016/j.jbiotec.2016.03.027

Figure Lengend Snippet: Fig. 2: High efficiency implementation of CAS9 and gRNA expression in P. pastoris.

Article Snippet: The Homo sapiens codon optimized CAS9 (HsCAS9) and the Streptococcus pyogenes CAS9 (SpCAS9) were amplified from the template vectors p414-TEF1p-Cas9-CYC1t and pMJ806 (both obtained from Addgene, Cambridge, MA, USA) using primer pairs spCas9_fw/spCas9-SV40_rv and hsCas9_fw/ hsCas9-SV40_rv respectively.

Techniques: Expressing

Journal: Journal of biotechnology

Article Title: Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris.

doi: 10.1016/j.jbiotec.2016.03.027

Figure Lengend Snippet: Fig. 4: The CRISPR/Cas9 system allows high efficiency targeting of various genes (A) and is suitable for multiplexing (B, C) in P. pastoris.

Article Snippet: The Homo sapiens codon optimized CAS9 (HsCAS9) and the Streptococcus pyogenes CAS9 (SpCAS9) were amplified from the template vectors p414-TEF1p-Cas9-CYC1t and pMJ806 (both obtained from Addgene, Cambridge, MA, USA) using primer pairs spCas9_fw/spCas9-SV40_rv and hsCas9_fw/ hsCas9-SV40_rv respectively.

Techniques: CRISPR, Multiplexing

Journal: Royal Society Open Science

Article Title: A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

doi: 10.1098/rsos.170095

Figure Lengend Snippet: Constitutive expression of Cas9 in L. mexicana. Growth curves of L. mexicana wild-type cells (WT) and L. mex Cas9 clone G2.

Article Snippet: For expression of Cas9 in Leishmania , plasmid pRM006 was constructed: humanized S. pyogenes Cas9 from plasmid pX330 (Addgene #42230; [ ]) was joined to Crithidia fasciculata PGKB 5′-UTR and GSPS 3′-UTR from pLENT V1 [ ] and the backbone of pLENT V1, containing a phleomycin-resistance gene, which was subsequently replaced with a hygromycin B phosphotransferase gene.

Techniques: Expressing

Journal: Royal Society Open Science

Article Title: A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

doi: 10.1098/rsos.170095

Figure Lengend Snippet: Short HF allow efficient integration of donor DNA. ( a ) Leishmania mexicana wild-type cells (WT; grey) and L. mex Cas9 clone G2 (red) were transfected with PF16::YFP tagging cassettes containing different length HF (24, 33, 47, 65 and 350 nt). The plot shows the number of transfectants recovered per transfected cell. Large open circles denote blasticidin-resistant cells; small-filled circles denote blasticidin-resistant cells with green fluorescent flagella. For wild-type cells, homology lengths of 24–65 nt yielded no drug-resistant cells. Each data point represents the mean number of transfectants from three independent transfections. ( b ) Micrographs showing PF16::YFP and PF16::mCherry (mCh) expression in L. mex Cas9 cells transfected with each tagging construct separately or combined. Merged: phase contrast image overlaid with fluorescence channels showing YFP (green), mCh (magenta) and Hoechst-stained DNA (cyan). Scale bar 5 µm.

Article Snippet: For expression of Cas9 in Leishmania , plasmid pRM006 was constructed: humanized S. pyogenes Cas9 from plasmid pX330 (Addgene #42230; [ ]) was joined to Crithidia fasciculata PGKB 5′-UTR and GSPS 3′-UTR from pLENT V1 [ ] and the backbone of pLENT V1, containing a phleomycin-resistance gene, which was subsequently replaced with a hygromycin B phosphotransferase gene.

Techniques: Transfection, Expressing, Construct, Fluorescence, Staining

Journal: Royal Society Open Science

Article Title: A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

doi: 10.1098/rsos.170095

Figure Lengend Snippet: Co-transfection of two PCR amplicons allowed precise insertion of marker genes. ( a ) PCR-amplified donor DNA containing 30 nt HF specific to the target locus, a fluorescent protein tag and a drug-selectable marker gene. ( b ) Strategy for sgRNA delivery: the sgRNA template is produced by PCR using an oligo encoding the T7 promoter, 20 nt defining the target-site and a sequence complementary to the 3′-end of the second oligo, comprising the sgRNA scaffold . The resulting PCR product is transfected into cells for T7 RNAP-driven transcription of the sgRNA. ( c ) Summary of outcome of transfections with different combinations of sgRNA templates and donor DNAs and electroporation protocols. Green filled circles denote drug-resistant cells showing the expected fluorescent signal; red open circles indicate failure to produce any drug-resistant transfectants. ( d ) Micrographs showing correct flagellar localization of PF16::YFP, nuclear localization of H2B::YFP (the white colour indicates co-localization of YFP and Hoechst) and flagellar membrane localization of SMP-1::YFP in cells that were co-transfected with the donor DNA and corresponding sgRNA template. Phase contrast image merged with mCh or YFP fluorescence channels and Hoechst-stained DNA (magenta). Scale bar 5 µm. ( e ) PCR-detection of the sgRNA template. Top, agarose gel showing the results of a diagnostic PCR to test for the presence of the sgRNA template. Template DNAs were as follows. Input: 1 µl of PF16 sgRNA PCR used for transfection; 5 min-48 h post transfection (p.t.): genomic DNA from cells at different time points post transfection with PF16 sgRNA; Δ PF16 , Δ LPG1 , PF16::mCh / PF16::YFP: genomic DNA from drug-resistant cell lines reported in this study; parental: genomic DNA from the parental cell line L. mex Cas9 T7. Bottom, diagram showing the sgRNA template and the primers used for PCR detection.

Article Snippet: For expression of Cas9 in Leishmania , plasmid pRM006 was constructed: humanized S. pyogenes Cas9 from plasmid pX330 (Addgene #42230; [ ]) was joined to Crithidia fasciculata PGKB 5′-UTR and GSPS 3′-UTR from pLENT V1 [ ] and the backbone of pLENT V1, containing a phleomycin-resistance gene, which was subsequently replaced with a hygromycin B phosphotransferase gene.

Techniques: Cotransfection, Marker, Amplification, Produced, Sequencing, Transfection, Electroporation, Membrane, Fluorescence, Staining, Agarose Gel Electrophoresis, Diagnostic Assay

Journal: Royal Society Open Science

Article Title: A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

doi: 10.1098/rsos.170095

Figure Lengend Snippet: A modular system for PCR-amplification of targeting fragments. ( a ) Strategy for using pT and pPLOT to generate donor DNA for repair of Cas9-induced double-strand breaks allowing precise modification of a target locus. To delete a target gene, two sgRNAs direct cuts to sites immediately upstream (5′) and downstream (3′) of the target gene. Repair cassettes with drug-selectable marker genes (DrugR) and 30 nt HF specific to the target locus are PCR-amplified from pT plasmids with primers 1 and 5. The same primer pair can be used to amplify cassettes with different drug-resistance genes. To tag a target gene, one sgRNA directs a cut immediately upstream or downstream of the target gene, for fusing tags to the N- or C- terminus of a protein, respectively. A repair cassette with 30 nt HF specific to the target locus, the desired tag and a drug-selectable marker gene are PCR-amplified from a pPLOT plasmid. Primer pair 1 and 2 is used for N-terminal tagging (indicated by grey arrows), 4 and 5 for C-terminal tagging (dashed arrows). The same primer pairs can be used to amplify a range of different tagging cassettes. Primers 3 and 6 (not shown) are used to amplify the 5′- and 3′-sgRNA templates. ( b ) Diagrams showing the target gene locus before and after insertion of repair cassettes.

Article Snippet: For expression of Cas9 in Leishmania , plasmid pRM006 was constructed: humanized S. pyogenes Cas9 from plasmid pX330 (Addgene #42230; [ ]) was joined to Crithidia fasciculata PGKB 5′-UTR and GSPS 3′-UTR from pLENT V1 [ ] and the backbone of pLENT V1, containing a phleomycin-resistance gene, which was subsequently replaced with a hygromycin B phosphotransferase gene.

Techniques: Amplification, Modification, Marker, Plasmid Preparation

Journal: Royal Society Open Science

Article Title: A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

doi: 10.1098/rsos.170095

Figure Lengend Snippet: Knockout of PF16. ( a ) PCR analysis of the Δ PF16 cell line. (i) PCR products visualized on agarose gel. P, parental cell line L. mex Cas9 T7; KO, Δ PF16 population; AB, cells expressing an ectopic copy of PF16 . (ii) Diagram showing the PF16 locus and PCR primers (arrows) used to test for presence of the PF16 CDS or the correct integration of the drug-resistance genes (blue boxes). ( b ) Transmission electron microscopy cross section showing the 9 + 2 microtubule arrangement in flagellar axonemes of the parental cell line, scale bar 100 nm. ( c–e ) Axonemes of Δ PF16 cells with a 9 + 2, 9 + 1 or 9 + 0 microtubule arrangement. ( f ) Measurements of the angle between the plane though the CP and the PFR; parental N = 23, Δ PF16 N = 21. ( g ) Cartoon illustrating how angles shown in ( f ) were measured.

Article Snippet: For expression of Cas9 in Leishmania , plasmid pRM006 was constructed: humanized S. pyogenes Cas9 from plasmid pX330 (Addgene #42230; [ ]) was joined to Crithidia fasciculata PGKB 5′-UTR and GSPS 3′-UTR from pLENT V1 [ ] and the backbone of pLENT V1, containing a phleomycin-resistance gene, which was subsequently replaced with a hygromycin B phosphotransferase gene.

Techniques: Knock-Out, Agarose Gel Electrophoresis, Expressing, Transmission Assay, Electron Microscopy

Journal: Royal Society Open Science

Article Title: A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

doi: 10.1098/rsos.170095

Figure Lengend Snippet: Knockout of LPG1. ( a,b ) PCR analysis of the Δ LPG1 cell line: test for the presence of the LPG1 CDS; ( c ) test for correct integration of the blasticidin-resistance gene; ( d ) test for correct integration of the neomycin-resistance gene, lanes as in ( c ). Diagrams above the gel pictures show the primers (arrows) used for PCR and size of expected product. ( e ) Western blot of whole-cell lysates probed with LT22. P, parental cell line L. mex Cas9 T7; KO POP, Δ LPG1 population; F3, Δ LPG1 clonal cell line; +AB, cell lines expressing an ectopic copy of LPG1 .

Article Snippet: For expression of Cas9 in Leishmania , plasmid pRM006 was constructed: humanized S. pyogenes Cas9 from plasmid pX330 (Addgene #42230; [ ]) was joined to Crithidia fasciculata PGKB 5′-UTR and GSPS 3′-UTR from pLENT V1 [ ] and the backbone of pLENT V1, containing a phleomycin-resistance gene, which was subsequently replaced with a hygromycin B phosphotransferase gene.

Techniques: Knock-Out, Western Blot, Expressing

Journal: Royal Society Open Science

Article Title: A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

doi: 10.1098/rsos.170095

Figure Lengend Snippet: Targeting GPI-PLC in T. brucei bloodstream forms. ( a ) Localization of tagged GPI-PLC in T. brucei SmOx B4 Cas9 cells co-transfected with sgRNA templates and donor DNA(s); one or both alleles of GPI-PLC were tagged with mNeonGreen (mNG) or TagRFP S158 T (, TagRFPt), as indicated. SmOx B4 is the parental untagged cell line. Scale bar 5 µm. ( b ) PCR analysis of the following cell lines: three independent Δ GPI-PLC clones, doubly tagged cell line mNG::GPI-PLC/TagRFPt::GPI-PLC, SmOx B4 pTB011 and SmOx B4. (i) PCR amplicons visualized on an agarose gel. (ii) diagrams showing the primers (arrows) used for PCR and size of expected product. ( c,d ) Cells were subjected to hypotonic lysis for 20 min, separated into pellet (p) and supernatant (s) fractions and run together with whole-cell lysates (w) on an SDS PAGE gel. ( c ) Western blot probed with anti-GPI-PLC (arrows indicate expected bands: TagRFPt::GPI-PLC, 67.5 kDa; mNG::GPI-PLC 66.5 kDa; GPI-PLC, 40 kDa); ( d ) Coomassie stained gel (arrow indicates VSG; m, 50 kDa marker).

Article Snippet: For expression of Cas9 in Leishmania , plasmid pRM006 was constructed: humanized S. pyogenes Cas9 from plasmid pX330 (Addgene #42230; [ ]) was joined to Crithidia fasciculata PGKB 5′-UTR and GSPS 3′-UTR from pLENT V1 [ ] and the backbone of pLENT V1, containing a phleomycin-resistance gene, which was subsequently replaced with a hygromycin B phosphotransferase gene.

Techniques: Transfection, Clone Assay, Agarose Gel Electrophoresis, Lysis, SDS Page, Western Blot, Staining, Marker

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