Journal: Cell Reports

Article Title: A Disease-Causing Single Amino Acid Deletion in the Coiled-Coil Domain of RAD50 Impairs MRE11 Complex Functions in Yeast and Humans

doi: 10.1016/j.celrep.2020.108559

Figure Lengend Snippet: P1’s Cells Exhibit a Reduced DNA End Resection and HDR (A–C) Immunofluorescence detection of P-KAP1 (A), RPA32 (B), and RAD51 (C) in primary fibroblasts from healthy control and P1 before and 6 h after 10 Gy irradiation. The image is representative of three independent experiments. (D) Quantitative analysis of RAD51 foci before and after IR in SV40-hTERT fibroblasts untransduced or transduced with an empty or WT-RAD50-expressing vector. The number of nuclei scored and statistical significances are noted. (E) Efficiency of HDR assessed by the Cas9-directed knockin of Clover in the Lamin-A (LMNA) coding sequence. Cells were transfected with the mCherry vector alone (used to control transfection efficiency) or in combination with the repair template sequence containing the Clover coding sequence in conjunction, or not, with the pX330-LMNA1 vector encoding Cas9 and the guide RNAs (gRNAs) targeting the LMNA sequence (denoted as CRISPR-Cas9). Relative HDR efficiency was measured by the percentage of Clover + cells triple transfected relative to cells transfected without the CRISPR-Cas9-gRNA-expressing vector. Results represents the mean and SD of triplicates from three independent experiments.

Article Snippet: 1 μg of mCherry vector (PGK-H2BmCherry, Addgene #21217), 4 μg of CRISPR/cas9 vector (pX330-LMNA-gRNA1, Addgene #122507) and 1 μg of donor vector (pCR2.1 Clover-LMNA donor, Addgene #122508) were used.

Techniques: Immunofluorescence, Irradiation, Transduction, Expressing, Plasmid Preparation, Knock-In, Sequencing, Transfection, CRISPR

Journal: Cell Reports

Article Title: A Disease-Causing Single Amino Acid Deletion in the Coiled-Coil Domain of RAD50 Impairs MRE11 Complex Functions in Yeast and Humans

doi: 10.1016/j.celrep.2020.108559

Figure Lengend Snippet:

Article Snippet: 1 μg of mCherry vector (PGK-H2BmCherry, Addgene #21217), 4 μg of CRISPR/cas9 vector (pX330-LMNA-gRNA1, Addgene #122507) and 1 μg of donor vector (pCR2.1 Clover-LMNA donor, Addgene #122508) were used.

Techniques: Recombinant, Fluorsave, Protease Inhibitor, Blocking Assay, Transfection, Mutagenesis, Transduction, Software

Journal: Cell Reports

Article Title: A Disease-Causing Single Amino Acid Deletion in the Coiled-Coil Domain of RAD50 Impairs MRE11 Complex Functions in Yeast and Humans

doi: 10.1016/j.celrep.2020.108559

Figure Lengend Snippet: P1’s Cells Exhibit a Reduced DNA End Resection and HDR (A–C) Immunofluorescence detection of P-KAP1 (A), RPA32 (B), and RAD51 (C) in primary fibroblasts from healthy control and P1 before and 6 h after 10 Gy irradiation. The image is representative of three independent experiments. (D) Quantitative analysis of RAD51 foci before and after IR in SV40-hTERT fibroblasts untransduced or transduced with an empty or WT-RAD50-expressing vector. The number of nuclei scored and statistical significances are noted. (E) Efficiency of HDR assessed by the Cas9-directed knockin of Clover in the Lamin-A (LMNA) coding sequence. Cells were transfected with the mCherry vector alone (used to control transfection efficiency) or in combination with the repair template sequence containing the Clover coding sequence in conjunction, or not, with the pX330-LMNA1 vector encoding Cas9 and the guide RNAs (gRNAs) targeting the LMNA sequence (denoted as CRISPR-Cas9). Relative HDR efficiency was measured by the percentage of Clover + cells triple transfected relative to cells transfected without the CRISPR-Cas9-gRNA-expressing vector. Results represents the mean and SD of triplicates from three independent experiments.

Article Snippet: 1 μg of mCherry vector (PGK-H2BmCherry, Addgene #21217), 4 μg of CRISPR/cas9 vector (pX330-LMNA-gRNA1, Addgene #122507) and 1 μg of donor vector (pCR2.1 Clover-LMNA donor, Addgene #122508) were used.

Techniques: Immunofluorescence, Irradiation, Transduction, Expressing, Plasmid Preparation, Knock-In, Sequencing, Transfection, CRISPR

Journal: Cell Reports

Article Title: A Disease-Causing Single Amino Acid Deletion in the Coiled-Coil Domain of RAD50 Impairs MRE11 Complex Functions in Yeast and Humans

doi: 10.1016/j.celrep.2020.108559

Figure Lengend Snippet:

Article Snippet: 1 μg of mCherry vector (PGK-H2BmCherry, Addgene #21217), 4 μg of CRISPR/cas9 vector (pX330-LMNA-gRNA1, Addgene #122507) and 1 μg of donor vector (pCR2.1 Clover-LMNA donor, Addgene #122508) were used.

Techniques: Recombinant, Fluorsave, Protease Inhibitor, Blocking Assay, Transfection, Mutagenesis, Transduction, Software

Journal: Molecular Therapy

Article Title: In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa

doi: 10.1038/mt.2015.220

Figure Lengend Snippet: Allele-specific targeting and disruption of Rho S334 in vitro . ( a ) Schematic of px330 construct used. ( b ) gRNA TRGT and predicted genomic DNA binding sites in Rho S334 and Rho WT . PAM (red underlined bases) and mismatches (red font) are shown. ( c ) Phase contrast photomicrograph of mCherry + and mCherry − MSCs S334 3-days post-lipofection with gRNA constructs prior to fluorescence-activated cell sorting (FACS) isolation. ( d ) FACS gating strategy for mCherry + MSC S334 isolation is shown. mCherry + cells represented 12% of the total population, with the brightest 3.4% mCherry + MSCs S334 selected for genomic DNA sequence analysis using PCR amplicons encompassing predicted Cas9 cleavage sites. ( e–h ) Genomic DNA sequencing results are shown with Phred quality scores (gray shading) at bottom. DNA disruption is shown from gRNA TRGT -transfected MSCs S334 downstream from the Cas9 cleavage site (blue highlight) in Rho S334 (e), but not in Rho WT (f). Genomic disruption was absent at the Rho S334 locus using gRNA CNTRL (g) or with no vector in untreated eyes (h). Bar = 400 µm. CMV, cytomegalovirus; MSC, mesenchymal stem cell; PAM, protospacer adjacent motif.

Article Snippet: Cas9/gRNA vector design. gRNAs were cloned into px330 vectors (Life Technologies, Carlsbad, CA) via BbsI restriction enzyme sites upstream of the scaffold gRNA sequence and the mCherry reporter (Addgene, Cambridge, MA) was cloned downstream of the Cas9 transcript, which was under constitutive expression by cytomegalovirus promoter.

Techniques: Disruption, In Vitro, Construct, Binding Assay, Fluorescence, FACS, Isolation, Sequencing, DNA Sequencing, Transfection, Plasmid Preparation

Journal: Molecular Therapy

Article Title: In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa

doi: 10.1038/mt.2015.220

Figure Lengend Snippet: Allele-specific targeting and disruption of Rho S334 in vivo . ( a ) Fast-Green DNA dye shows plasmid distribution following unilateral subretinal injection in S334ter-3 rats at P0. ( b ) Representative retinal flat-mount shows variable mCherry intensity and uneven distribution 4 days after injection. ( c ) Gating strategy for fluorescence-activated cell sorting isolation of enzymatically dissociated P4 retinal cells with mCherry fluorescence intensity at high (red, 0.3%) intermediate (purple, 0.6%), and no (blue, 97.9%) expression. Sanger sequencing of PCR-amplified genomic Rho loci from photoreceptors showed in vivo disruption of Rho S334 ( d ), but not Rho WT ( e ) using gRNA TRGT . ( f ) Rho S334 locus targeting schematic and deep sequencing reads shows insertions/deletions (indels) proximal to the predicted cleavage site (arrowhead) from gRNA TRGT -expressing cells. PAM, protospacer adjacent motif.

Article Snippet: Cas9/gRNA vector design. gRNAs were cloned into px330 vectors (Life Technologies, Carlsbad, CA) via BbsI restriction enzyme sites upstream of the scaffold gRNA sequence and the mCherry reporter (Addgene, Cambridge, MA) was cloned downstream of the Cas9 transcript, which was under constitutive expression by cytomegalovirus promoter.

Techniques: Disruption, In Vivo, Plasmid Preparation, Injection, Fluorescence, FACS, Isolation, Expressing, Sequencing, Amplification

Journal: Molecular Therapy

Article Title: In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa

doi: 10.1038/mt.2015.220

Figure Lengend Snippet: Phenotypic rescue by Rho S334 -selective ablation . ( a–e ) Fluorescent confocal images of gRNA TRGT -treated ( a – c ) and gRNA CNTRL -treated ( d , e ) retinas at P33. ( a ) Montage image shows that mCherry distribution correlated with ONL rescue (DAPI, blue) and POS formation (RHO WT C-terminal immunolabel, green). Inset: magnified image of outlined region shows preserved ONL with organized POS (arrowheads) adjoining degenerated ONL with diminished POS (arrows). ( b , c ) RHO N-terminal ( b ) and C-terminal ( c ) immunostaining in gRNA TRGT -treated retinas was absent from PR cell bodies and localized to OS. Significant PR preservation in the ONL was observed from gRNA TRGT treatment ( b , bracketed areas). ( d , e ) RHO N-terminal ( d ) and C-terminal ( e ) immunostaining was absent in gRNA CNTRL -treated retinas, which lacked POS formation and ONL contained one row of remaining PR nuclei ( d , bracketed areas). DAPI, 49,69-diamidino-2-phenylindole; INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OS, outer segment; POS, photoreceptor outer segment; PR, photoreceptor.

Article Snippet: Cas9/gRNA vector design. gRNAs were cloned into px330 vectors (Life Technologies, Carlsbad, CA) via BbsI restriction enzyme sites upstream of the scaffold gRNA sequence and the mCherry reporter (Addgene, Cambridge, MA) was cloned downstream of the Cas9 transcript, which was under constitutive expression by cytomegalovirus promoter.

Techniques: Immunolabeling, Immunostaining, Preserving

Journal: Molecular Therapy

Article Title: In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa

doi: 10.1038/mt.2015.220

Figure Lengend Snippet: Rho S334 ablation preserved cone morphology and second-order retinal neuron synapses . Fluorescence confocal microscopy images from gRNA CNTRL -treated ( a–d ) or gRNA TRGT -treated ( e–j ) eyes at P33. ( a , b ) Surviving PRs after gRNA CNTRL treatment were non- Rho -expressing cones PRs ( a , d , cone arrestin, green), which lacked typical morphological features observed in retinas rescued with gRNA TRGT treatment ( e , h ). Individual channel images corresponding to bracketed areas in ( a ) and ( e ) show rescued PR nuclei (DAPI, ( b ) versus ( f )) in gRNA vector transfected areas (mCherry, ( c ) versus ( g )) with preserved cone morphology ( i.e. , pedicles and POS, ( d ) versus ( h )). ( i ) Greater dendritic arborization of INL-resident rod-bipolar neurons (PKC-α, green) was evident at the OPL in mCherry + areas following gRNA TRGT treatment (left inset), in contrast to the adjacent degenerated area lacking mCherry + (right inset). ( j ) Similarly, synaptophysin immunolabel (green) showed greater intensity in OPL regions in which PR nuclei preservation (DAPI) corresponded with mCherry + expression (inset: arrowheads), in sharp contrast with the adjacent unprotected area to which gRNA TRGT transfection did not extend (inset: arrows). DAPI, 49,69-diamidino-2-phenylindole; INL, inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer; PKC-α, protein kinase C-alpha; PR, photoreceptor.

Article Snippet: Cas9/gRNA vector design. gRNAs were cloned into px330 vectors (Life Technologies, Carlsbad, CA) via BbsI restriction enzyme sites upstream of the scaffold gRNA sequence and the mCherry reporter (Addgene, Cambridge, MA) was cloned downstream of the Cas9 transcript, which was under constitutive expression by cytomegalovirus promoter.

Techniques: Fluorescence, Confocal Microscopy, Expressing, Plasmid Preparation, Transfection, Immunolabeling, Preserving

Journal: Molecular Therapy

Article Title: In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa

doi: 10.1038/mt.2015.220

Figure Lengend Snippet: PR rescue by gRNA TRGT treatment corresponded with vision rescue . ( a ) Fluorescent microscopy montage image shows mCherry + reporter distribution (arrows) of gRNA TRGT vector in a retinal flat-mount at P33 calculated at 29% of total retina area by NIH ImageJ analysis. ( b ) By retinal cross-section, mCherry + regions from gRNA TRGT treatment contained significantly more PR nuclei than the mCherry + regions from gRNA CNTRL treatment, or comparable regions from untreated control areas (gRNA TRGT : 307 ± 82 PR nuclei/100 µm, N = 5 versus gRNA CNTRL : 33 ± 3, ** P ≤ 0.01, N = 3 versus Untreated: 27 ± 13, †† P ≤ 0.01, N = 4). ( c ) Visual acuity (optokinetic response) was significantly higher from gRNA TRGT treatment at P39, than from gRNA CNTRL treatment (gRNA TRGT : 0.185 ± 0.008 c/d, N = 5 versus gRNA CNTRL : 0.121 ± 0.009 c/d, N = 4, †† P ≤ 0.01). Visual acuity in gRNA TRGT -treated eyes was significantly higher than in untreated contralateral eyes (Treated: 0.185 ± 0.008 versus Contralateral: 0.138 ± 0.006 c/d, N = 5, *** P ≤ 0.001). Visual acuity in eyes injected with gRNA CNTRL was not different from that of contralateral noninjected eyes (Treated: 0.121 ± 0.009 versus Contralateral: 0.121 ± 0.012 c/d, N = 4, P = 0.763). ( d ) By using the fellow eyes of individual animals as internal controls, the higher visual acuity from gRNA TRGT treatment represented a 35 ± 4.6% increase, compared to a 2.3 ± 0.7% decrease with gRNA CNTRL injection ( P < 0.01). c/d = cycles/degree. All values represent mean ± SEM. N.S., not significant; PR, photoreceptor.

Article Snippet: Cas9/gRNA vector design. gRNAs were cloned into px330 vectors (Life Technologies, Carlsbad, CA) via BbsI restriction enzyme sites upstream of the scaffold gRNA sequence and the mCherry reporter (Addgene, Cambridge, MA) was cloned downstream of the Cas9 transcript, which was under constitutive expression by cytomegalovirus promoter.

Techniques: Microscopy, Plasmid Preparation, Control, Injection