grna expression empty vector lentiguide puro  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

doi: 10.1093/nar/gkw179

Figure Lengend Snippet: HR repair of nicks on the lagging-strand template is promoted by replication. ( A ) The DR-GFP reporter consists of two inactive copies of the GFP gene. Induction of a DSB or nick in SceGFP triggers HR that uses iGFP as a template to restore the GFP open reading frame. The inset shows the SceGFP sequence around the 18 bp I-SceI recognition site, which begins at the in-frame stop codon (red). PAMs required for Cas9 binding are shaded gray and the sequences bound by the indicated gRNAs are shaded in light blue (gRNA01) and light purple (gRNA02). Cas9 variants: wild-type (Cas9 WT ), D10A (Cas9 D ) and H840A (Cas9 H ). The predicted Cas9 cleavage sites are marked by colored triangles. T - transcribed strand, N - non-transcribed strand. ( B–D ) U2OS-DR-GFP cells (B and C) or ES-DR-GFP cells (D) were transfected with expression vectors for the indicated Cas9 and gRNA variants, or I-SceI. As a negative control (first bar in graphs B–D), Cas9 WT was transfected with a gRNA expression vector that lacked the targeting portion of the gRNA sequence. ( E ) U2OS-DR-GFP cells were arrested at the G 2 /M phase transition by 16 h incubation in 500 nM nocodazole or mock-treated with DMSO, stained with propidium iodide and analyzed by flow cytometry to confirm the cell cycle arrest. The left panel shows representative histograms of DNA content. The right panel shows the average percentage of cells in the indicated cell cycle phases. ( F ) U2OS-DR-GFP cells were treated as in (E) and transfected with the indicated Cas9 and gRNA expression vectors, then mock-treated or incubated in the presence of nocodazole for a further 24 h and analyzed by flow cytometry. The cell cycle distribution of cells at the time of flow cytometric analysis is shown in Supplementary Figure S1I. ( G ) Schematic overview of the DR-Orip-GFP reporter. EBNA1 initiates replication downstream from SceGFP , such that the top strand becomes the lagging-strand template. The inset depicts the predicted sites of cleavage by Cas9 D (filled triangles) or Cas9 H (empty triangles), in combination with the indicated gRNAs. ( H ) U2OS cells were transfected with the DR-OriP-GFP reporter, the empty vector (+ Vector) or EBNA1 expression vector (+ EBNA1), gRNA01 and the indicated Cas9 variant. Cas9 D nicks the leading-strand template and Cas9 H nicks the lagging-strand template, as indicated. ( I ) Cells were transfected as in (H), except gRNA02 was used, such that Cas9 D nicks the lagging-strand template and Cas9 H the leading-strand template.

Article Snippet: Cas9 WT , Cas9 D and empty gRNA expression vectors were purchased from Addgene (ID 41815, 41816 and 41824) ( ).

Techniques: Sequencing, Binding Assay, Transfection, Expressing, Negative Control, Plasmid Preparation, Sublimation, Incubation, Staining, Flow Cytometry, Variant Assay

Journal: Nucleic Acids Research

Article Title: Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

doi: 10.1093/nar/gkw179

Figure Lengend Snippet: nick HR requires established HR factors but is not suppressed by NHEJ components. ( A and B ) U2OS-DR-GFP cells transfected with the indicated Cas9, I-SceI and gRNA expression vectors were incubated for 48 h in the presence of the various concentrations of ATM inhibitor KU55933 (A) or ATR inhibitor VE-821 (B). ( C ) Schematic representation of HR and its steps that are inhibited by the BRCA1-interacting peptide hB202 (from BARD1) and the RAD51-interacting peptide BRC3 (from BRCA2). ( D ) U2OS-DR-GFP cells were transfected with the indicated Cas9 and gRNA or I-SceI expression vectors and either with the empty expression vector (pCAGGS) or with the vectors encoding the hB202 and BRC3 peptides. ( E ) Canonical NHEJ is known to suppress DSB HR, but its effect on nick HR is uncertain. ( F ) Wild-type (J1), Ku70 −/− or Xrcc4 −/− ES-DR-GFP cells were transfected with the indicated expression vectors. ( G ) Ku70 −/− ES-DR-GFP cells were transfected with the indicated expression vectors and either the empty (+ Vector) or KU70 (+ KU70) expression vector. ( H ) Wild-type (E14) or Dna-Pk −/− ES-DR-GFP cells were transfected with the indicated expression vectors. ( I ) U2OS-DR-GFP cells were transfected and analyzed as in (A and B), but incubated in the presence of various concentrations of a DNA-PK inhibitor (NU7441).

Article Snippet: Cas9 WT , Cas9 D and empty gRNA expression vectors were purchased from Addgene (ID 41815, 41816 and 41824) ( ).

Techniques: Transfection, Expressing, Incubation, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

doi: 10.1093/nar/gkw179

Figure Lengend Snippet: Paired nicks induce intrachromosomal HR. ( A ) Schematic of paired nicks with the potential to generate 5′ or 3′ overhangs to be tested for induction of PN HR. ( B ) Reporters for measuring PN HR between repeats. The left panel shows a schematic of the pnDR-GFP20-940 bp reporters. The inset shows the relative binding positions of the gRNAs (marked by the green or black horizontal bars) and Cas9 cleavage sites (dotted vertical lines). The lengths of the 5′ or 3′ nick offset as well as the heterology (orange bars) are indicated. The right panel shows expected cleavage positions in the pnDR-GFP reporter with the indicated gRNA/Cas9 combinations. ( C-E ) U2OS cells were transiently transfected with the indicated pnDR-GFP reporter and Cas9/gRNA expression vectors required to generate single or paired nicks or DSBs (schematically drawn under each bar). ( F-H ) ES cells with the various pnDR-GFP reporters integrated at the Hprt locus were transfected with the indicated Cas9 and gRNA expression vectors to generate single or paired nicks or DSBs (schematically drawn under each bar). ( I ) Schematic representation of the pnDR-GFP0 bp reporter, analogous to (B). ( J ) U2OS cells were transfected with the pnDR-GFP0 bp reporter and the indicated Cas9 and gRNA expression vectors. ( K ) ES cells with pnDR-GFP0 bp integrated at the Hprt locus were transfected with the indicated Cas9 and gRNA expression vectors.

Article Snippet: Cas9 WT , Cas9 D and empty gRNA expression vectors were purchased from Addgene (ID 41815, 41816 and 41824) ( ).

Techniques: Binding Assay, Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

doi: 10.1093/nar/gkw179

Figure Lengend Snippet: Ku suppression of PN HR depends on paired nick offset type and distance. ( A ) Schematic representation of the distinct genetic control of HR triggered by different DNA lesions. The NHEJ component Ku suppresses DSB and PNHR resulting from 5′ overhangs but does not affect nickHR or PNHR resulting from 3′ overhangs. ( B and C ) Ku70 −/− ES-pnDR-GFP-50 bp (B) or Ku70 −/− ES-pnDR-GFP-0 bp (C) cells were transfected with the indicated Cas9 and gRNA expression vectors required to generate single or paired lesions (drawn schematically under each bar) and either the empty (+ Vector) or KU70 (+ KU70) expression vector. ( D–F ) Ku70 −/− ES cells with the various pnDR-GFP reporter variants were transfected with the expression vectors for Cas9 WT (D), Cas9 D (E) or Cas9 H (F), gRNA09enh, gRNA10enh and either the empty (+ Vector) or KU70 (+ KU70) expression vector.

Article Snippet: Cas9 WT , Cas9 D and empty gRNA expression vectors were purchased from Addgene (ID 41815, 41816 and 41824) ( ).

Techniques: Control, Transfection, Expressing, Plasmid Preparation