mlui hf  (New England Biolabs)

Journal: bioRxiv

Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

doi: 10.1101/243683

Figure Lengend Snippet: Representative western blots of retinal SpCas9 expression. (A) SpCas9 and YFP expression in the mouse retinas were assessed by western blot analysis. (B) Uncropped western blot images.Three retinas from different mice in each group were randomly selected for western blot analysis.

Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

Techniques: Western Blot, Expressing, Mouse Assay

Journal: bioRxiv

Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

doi: 10.1101/243683

Figure Lengend Snippet: Long-term effect of AAV2-mediated CRISPR/Cas administration on retinal function. Averaged ERG waveforms at selected intensities for control (black traces) and SpCas9 sgRNA/YFP sgRNA2 (n= 4, red traces; A), SpCas9 sgRNA/LacZ sgRNA (n = 4, blue traces; C) and YFP sgRNA2 (n= 5, green traces; E) injected eyes. Group average (± SEM) photoreceptoral (a-wave), bipolar cell (b-wave), amacrine cell (oscillatory potentials, OPs) and ganglion cell (scotopic threshold response, STR) amplitude relative to contralateral control eyes (%) for each group (B, D and F). Effect of SpCas9 sgRNA/YFP sgRNA2, SpCas9 sgRNA/LacZ sgRNA and YFP sgRNA2 on retinal structure measured with OCT (G). Group average (± SEM) retinal nerve fibre layer thickness (H) for SpCas9 sgRNA/YFP sgRNA2 treated (filled red, n= 4) and their contralateral controls (unfilled red, n= 4), SpCas9 sgRNA/LacZ sgRNA treated (filled blue, n= 4) and their contralateral controls (unfilled blue, n= 4) and YFP sgRNA2 treated (filled green, n= 5) and their contralateral controls (unfilled green, n=5). Total retinal thickness (I). Statistical analysis between injected and control eyes was performed using two-tailed Student t-test (*p

Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

Techniques: CRISPR, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

doi: 10.1101/243683

Figure Lengend Snippet: In vitro validation of kamikaze CRISPR/Cas construct. (A) Schematic of plasmid constructs for in vitro validation. (B) Representative Western blots of SpCas9 protein expression in cells co-transfected with SpCas9 and kamikaze (SpCas9 sgRNA/YFP sgRNA and SpCas9 sgRNA/LacZ sgRNA) or non-kamikaze (YFP sgRNA and LacZ sgRNA) constructs. (C) Representative images of YFP expression in cells co-transfected with kamikaze (SpCas9 sgRNA/YFP sgRNA and SpCas9 sgRNA/LacZ sgRNA) or non-kamikaze (YFP sgRNA and LacZ sgRNA) constructs. Percentage YFP disruption was assessed by FACS at 10 day after transfection. scale bar: 100 μm. Mean ± SEM for 2 independent replicates.

Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

Techniques: In Vitro, CRISPR, Construct, Plasmid Preparation, Western Blot, Expressing, Transfection, FACS

Journal: bioRxiv

Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

doi: 10.1101/243683

Figure Lengend Snippet: YFP sgRNA2 alone affects inner retinal function. (A) Averaged ERG waveforms at selected intensities for control (n=5, black) and treated eyes (n=5, green). (B) Groups average (±SEM) photoreceptoral (a-wave) saturated amplitude for contralateral control (unfilled) and treated eyes (filled). (C) Photoreceptoral sensitivity to light. (D) Intensity response characteristics across the entire range of intensities. (E) Bipolar cell amplitude. (F) Bipolar cell sensitivity to light. (G) Inner retinal amacrine cell mediated response (oscillatory potentials). Data are expressed as the Mean ± SEM. Statistical analysis between groups was performed using using two-tailed Student’s t-test.

Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

doi: 10.1101/243683

Figure Lengend Snippet: Kamikaze CRISPR/Cas-mediated genome editing of retinal cells in vivo . (A) High magnification of retinal flat-mount images, showing differences in YFP expression following AAV2-mediated delivery of SpCas9 sgRNA/YFP sgRNA (n= 5), YFP sgRNA2 (n= 5) or SpCas9 sgRNA/LacZ sgRNA (n= 3). scale bar: 20 μm. (B) Percentage YFP disruption was assessed by manual cell counting. Mean ± SEM for 3-5 independent replicates. Statistical analysis between groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test (**p

Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

Techniques: CRISPR, In Vivo, Expressing, Cell Counting

Journal: bioRxiv

Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

doi: 10.1101/243683

Figure Lengend Snippet: Uncropped agarose gel and western blot images. (A) In situ test of designed SpCas9 sgRNAs. (B) in vitro validation of designed SpCas9 sgRNAs. (C) Time course of SpCas9 expression. (D) in vitro validation of YFP targeting kamikaze CRISPR constructs. The β-actin membrane was reprobing without stripping.

Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

Techniques: Agarose Gel Electrophoresis, Western Blot, In Situ, In Vitro, Expressing, CRISPR, Construct, Stripping Membranes

Journal: bioRxiv

Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

doi: 10.1101/243683

Figure Lengend Snippet: Quantification of YFP disruption in the retina. The percentage of YFP disruption following AAV2-mediated delivery of SpCas9 sgRNA/YFP sgRNA6, YFP sgRNA6 or SpCas9 sgRNA/LacZ sgRNA was assessed by manual cell counting. Representative data are shown for 3-5 retinas and expressed as the Mean ± SEM. Statistical analysis between groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test (*p

Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

Techniques: Cell Counting

Journal: bioRxiv

Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

doi: 10.1101/243683

Figure Lengend Snippet: Time course of SpCas9 mRNA expression in the mouse retina. SpCas9 mRNA were isolated from the mice retina administrated with AAV2-SpCas9 sgRNA/YFP sgRNA2 or AAV2-YFP sgRNA2 at 5, 6 and 8 weeks after intravitreal injection. Relative fold change of SpCas9 expression was normalized by week 1 from each treatment group. Representative data are shown for 5-6 retinas per group/time point and expressed as Mean ± SEM. Statistical analysis between groups was performed using Two-way ANOVA followed by Sidak's multiple comparisons test, a , YFP sgRNA2: 1 vs 8 weeks, p=0.002. b , SpCas9/YFP sgRNA: 1 vs 8 weeks, p =0.7043. c , YFP sgRNA2 vs SpCas9/YFP sgRNA, p=0.0142.

Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

Techniques: Expressing, Isolation, Mouse Assay, Injection

Journal: bioRxiv

Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

doi: 10.1101/243683

Figure Lengend Snippet: Schematics of Kamikaze CRISPR/Cas system. A dual AAV vector system was used. One viral vector was used to deliver SpCas9 and the other delivered sgRNAs against SpCas9 and the target locus (YFP), in the presence of mCherry.

Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

Techniques: CRISPR, Plasmid Preparation

Journal: bioRxiv

Article Title: Using a reverse genetics system to generate recombinant SARS-CoV-2 expressing robust levels of reporter genes

doi: 10.1101/2022.05.21.492922

Figure Lengend Snippet: Assembly of entire SARS-CoV-2 genome into a BAC. a. The released five fragments (F1-F5) from pUC57 plasmids. b. Colony PCR screening for positive pBeloBAC-F1. c. Restriction digestion analysis of pBeloBAC-F13 using PacI and MluI. d. Restriction digestion analysis of pBeloBAC-F134 using PacI and MluI (F3); and MluI and BstBI (F4). e. Restriction digestion analysis of pBeloBAC-F1342 using KasI and PacI (F2); PacI and MluI (F3); and MluI and BstBI (F4). f. Restrict digestion confirmation of the pBeloBAC-FL using KasI and PacI (F2); PacI and MluI (F3); MluI and BstBI (F4); and BstBI and BamHI (F5).

Article Snippet: MluI-HF (New England Biolabs, catalog #: R3198L).

Techniques: BAC Assay, Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Using a reverse genetics system to generate recombinant SARS-CoV-2 expressing robust levels of reporter genes

doi: 10.1101/2022.05.21.492922

Figure Lengend Snippet: Recovery of rSARS-CoV-2 from the pBeloBAC-FL. a. Protocol used for recovery of rSARS-CoV-2 from the pBeloBAC-FL using Lipofectamine 2000 transfection in Vero E6 cells. b. CPE caused by rSARS-CoV-2/WT in Vero E6 cells. Scale bars, 100 µm. c. Confirmation of the rescued rSARS-CoV-2/WT by IFA using a mouse antibody against viral N protein and a TRITC labeled donkey anti-mouse IgG secondary antibody. The nucleus was stained by DAPI. d. Sanger sequencing of the M gene of the natural isolate SARS-CoV-2 (top) and the recombinant SARS-CoV-2(bottom) that the MluI was removed by silent mutation.

Article Snippet: MluI-HF (New England Biolabs, catalog #: R3198L).

Techniques: Transfection, Immunofluorescence, Labeling, Staining, Sequencing, Recombinant, Mutagenesis

Journal: bioRxiv

Article Title: Using a reverse genetics system to generate recombinant SARS-CoV-2 expressing robust levels of reporter genes

doi: 10.1101/2022.05.21.492922

Figure Lengend Snippet: Overview of a BAC-based RG system for generation of rSARS-CoV-2. a. The schematic representation of the SARS-CoV-2 genome. Unique restriction sites used for vial genome assembly are indicated. b. Commercially synthesized five fragments (F1-F5) in pUC57 plasmids. Restriction sites used to release each of the viral fragments are indicated. The MluI and BstBI highlighted in red are the restriction sites that have been removed by silent mutation and used as genetics tags. c. Assembly of the entire SARS-CoV-2 genome into the empty pBeloBAC to generate the full-length rescue plasmid (pBeloBAC-FL).

Article Snippet: MluI-HF (New England Biolabs, catalog #: R3198L).

Techniques: BAC Assay, Synthesized, Mutagenesis, Plasmid Preparation