Journal: Nature

Article Title: A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases

doi: 10.1038/s41586-019-1786-y

Figure Lengend Snippet: Plaque assay with the indicated phage spotted in ten-fold serial diluations on a lawn of P. aeruginosa , dark clearings in the lawn represent phage replication. Strain PAO1 expressing: a, Type I-C cas genes ( cas3-5-8-7 ) and crRNAs targeting the indicated phages. b, Type I-C cas genes and crRNAs targeting phage JBD30 or distinct phage ΦKZ-targeting crRNAs (#1-#3). c, Type II-A cas9 and distinct single guide RNAs (sgRNAs) targeting the indicated phage. d, Type V-A cas12a and distinct crRNAs against the indicated phage. e, Endogenous Type I R-M system ( hsdRSM ) in strain PAO1 was assayed using phages propagated on PAK (e.g. JBD30•PAK was first propagated on strain PAK). Together with an isogenic PAO1 ∆hsdR knockout. f, Type II EcoRI R-M system. Restriction activity was assayed using phages JBD30 and ΦKZ. All plaque assays replicated ≥ 2 times with similar results.

Article Snippet: The wild type allele of the M. EcoRI (Methyltransferase) and R.EcoRI (Endonuclease) genes were sub-cloned from pSB1A3 EcoRI Methylase-AmilCP and pSB1A3 EcoRI-RTX with EcoRI Methylase-AmilCP (Addgene plasmid # 85166, Addgene plasmid # 85165)p2CT-His-MBP-Lse_C2c2_WT and p2CT-His-MBP-Lsh_C2c2_WT (Addgene) into pHERD30T using Gibson assembly.

Techniques: Plaque Assay, Expressing, Knock-Out, Activity Assay

Journal: Nature

Article Title: A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases

doi: 10.1038/s41586-019-1786-y

Figure Lengend Snippet: a , ΦKZ and JBD30 genomic DNA digested with the indicated restriction enzymes in vitro . The first lane contains a 1 kb DNA ladder. The number of cut sites for each enzyme is shown at the bottom of the gels. Products were visualized on a 0.7% agarose gel, visualized with SYBR Safe nucleic acid stain. b, ΦKZ phage genomic DNA digested in vitro using KasI, and incubated with and without Cas9 loaded with crRNA:tracrRNA targeting the fragment liberated by KasI. Products were visualized on a 0.7% agarose gel, visualized with SYBR Safe nucleic acid stain. c, Strain PAO1 expressing EcoRI-Cherry, EcoRI-Cherry-ORF152, or EcoRI E111G-Cherry-ORF152 fusion protein. Plaque assays were conducted as in and quantified (n=3). Mean values are plotted as bars with error bars representing standard deviation. A t-test comparing ΦKZ EOP on PAO1 pEcoRI-Cherry to PAO1 pEcoRI-Cherry-ORF152 yielded a p-value of 2.88×10 −4 . d, Growth curves monitoring the OD 600 of PAO1 cells infected with the indicated ΦKZ multiplicity of infection (MOI). e, Live fluorescence imaging of infected P. aeruginosa strains engineered to express EcoRI-Cherry, EcoRI E111G-Cherry, EcoRI-Cherry-ORF152 or EcoRI E111G-Cherry-ORF152. DAPI stain shows the phage DNA. Cherry shows EcoRI fusion protein. In vitro digestion experiments a and b replicated twice with similar results. Plaque assays ( c ), growth curves ( d ), and microscopy ( e ) were replicated ≥ 3 times.

Article Snippet: The wild type allele of the M. EcoRI (Methyltransferase) and R.EcoRI (Endonuclease) genes were sub-cloned from pSB1A3 EcoRI Methylase-AmilCP and pSB1A3 EcoRI-RTX with EcoRI Methylase-AmilCP (Addgene plasmid # 85166, Addgene plasmid # 85165)p2CT-His-MBP-Lse_C2c2_WT and p2CT-His-MBP-Lsh_C2c2_WT (Addgene) into pHERD30T using Gibson assembly.

Techniques: In Vitro, Agarose Gel Electrophoresis, Staining, Incubation, Expressing, Standard Deviation, Infection, Fluorescence, Imaging, Microscopy

Journal: Nature

Article Title: A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases

doi: 10.1038/s41586-019-1786-y

Figure Lengend Snippet: a, Strain PAO1 was engineered to express EcoRI or EcoRI-ORF152 fusion protein. Plaque assays were conducted as in and quantified (n=3). b, Growth curves were conducted, monitoring the OD 600 of PAO1 cells infected with the indicated ΦKZ multiplicity of infection (MOI). c, Live fluorescence imaging of P. aeruginosa strains engineered to express EcoRI or EcoRI-ORF152. DAPI stain shows the phage DNA. d, Live fluorescence imaging of P. aeruginosa strains engineered to express EcoRI, or EcoRI-ORF152. DAPI stain shows the phage DNA within the shell. Wide field of view. All plaque assays were replicated 3 times with similar results. Growth curve experiments were replicated twice with similar results. Microscopy was replicated twice with similar results.

Article Snippet: The wild type allele of the M. EcoRI (Methyltransferase) and R.EcoRI (Endonuclease) genes were sub-cloned from pSB1A3 EcoRI Methylase-AmilCP and pSB1A3 EcoRI-RTX with EcoRI Methylase-AmilCP (Addgene plasmid # 85166, Addgene plasmid # 85165)p2CT-His-MBP-Lse_C2c2_WT and p2CT-His-MBP-Lsh_C2c2_WT (Addgene) into pHERD30T using Gibson assembly.

Techniques: Infection, Fluorescence, Imaging, Staining, Microscopy

Journal: Nature

Article Title: A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases

doi: 10.1038/s41586-019-1786-y

Figure Lengend Snippet: a, PAO1 expressing LseCas13a and a crRNA targeting phage ΦKZ. Plaque assays were conducted as in . b, Growth curves measuring the OD 600 of PAO1 infected with the indicated ΦKZ MOI. c, Cas13 escaper ΦKZ phage mutant deletions (red dashed lines) with target sites indicated (blue line). d, Live fluorescence imaging of P. aeruginosa strains engineered to express LseCas13a and crRNAs targeting ΦKZ. Cyan stain shows the phage DNA. e , Model summarizing the ΦKZ nucleus-like structure excluding (flat arrow) Cas9, Cas12, Cascade-Cas3 (Type I-C, Type I-F) and Type I restriction endonucleases (REase) and Type II REase, while the mRNA (red) is exported and can be targeted by Cas13. The nucleus-like structure is resistant to the indicated nucleases, but EcoRI fusion (to internal protein ORF152) enables targeting. Cas13 plaque assays a were replicated >3 times with similar results. Growth curve experiments b were replicated twice with similar results. Escaper mutants c were isolated once and verified by PCR, sequencing, and plaque assays. Microscopy d was replicated twice with similar results.

Article Snippet: The wild type allele of the M. EcoRI (Methyltransferase) and R.EcoRI (Endonuclease) genes were sub-cloned from pSB1A3 EcoRI Methylase-AmilCP and pSB1A3 EcoRI-RTX with EcoRI Methylase-AmilCP (Addgene plasmid # 85166, Addgene plasmid # 85165)p2CT-His-MBP-Lse_C2c2_WT and p2CT-His-MBP-Lsh_C2c2_WT (Addgene) into pHERD30T using Gibson assembly.

Techniques: Expressing, Infection, Mutagenesis, Fluorescence, Imaging, Staining, Isolation, Sequencing, Microscopy

Journal: mBio

Article Title: Genome-Wide Profiling of Cervical RNA-Binding Proteins Identifies Human Papillomavirus Regulation of RNASEH2A Expression by Viral E7 and E2F1

doi: 10.1128/mBio.02687-18

Figure Lengend Snippet: RNASEH2A is regulated by viral E7 via E2F1 and its expression in association with E2F1 and PCNA in cervical and endocervical cancer and head and neck squamous cell carcinoma. (A) Read coverage maps showing the RNA expression levels of all 7 cervical cancer samples and 7 normal samples for RNASEH2A. Distributions of RNASEH2A reads (blue for normal, red for cancer) along with RNASEH2A transcript structure were visualized using the Integrative Genomics Viewer (IGV). (B) Correlation between RNASEH2A and HPV16 E7 mRNAs in 48 clinical cervical lesion samples, including 25 CIN2-CIN3 and 23 cervical cancer tissue samples. (C) siRNA-specific knockdown of HPV16 or HPV18 E7 expression in CaSki (HPV16 + ) cells or HPV18-immortalized human foreskin keratinocytes (HFK18) with respect to expression of RNASEH2A, PCNA, and NOVA1 was evaluated by Western blotting. si-NS, nonspecific siRNA; si-E7, HPV16 or HPV18 E7-specific siRNA. Knockdown efficiency of E7 expression was evaluated by analysis of increased expression of p53 due to the E7 siRNA also targeting the overlapped bicistronic E6 RNA ; tubulin served as a sample loading control. (D) siRNA-specific knockdown of HPV16 or HPV18 E6 or E7 in CaSki (HPV16 + ) or HeLa (HPV18 + ) cells with respect to RNASEH2A expression was evaluated by TaqMan RT-qPCR. si-NS, nonspecific siRNA; si-E6, HPV16 or HPV18 E6-specific siRNA; si-E7, HPV16 or HPV18 E7-specific siRNA. (E and F) Specific-siRNA knockdown (E) or ectopic expression of E2F1 (F) in CaSki or HeLa cells with respect to RNASEH2A expression was evaluated by TaqMan RT-qPCR. si-NS, nonspecific siRNA; si-E2F1, E2F1-specific siRNA; p, control vector; p-E2F1, E2F1-expression vector. * , P < 0.05; * * , P < 0.01; ** * , P < 0.001. The relative expression levels of RNASEH2A in panels D, E, and F are shown as means ± SD for each treatment group determined in duplicate from three independent experiments, with the level in the si-NS (D and E) or vector control (F) group being set to 1. (G) Increased coexpression of RNASEH2A, PCNA, and E2F1 in cervical squamous cell carcinoma, endocervical adenocarcinoma (CESC), and head and neck squamous cell carcinoma (HNSC). Data were extracted from the TCGA data sets using TCGA2STAT R package version 1.2 ( https://CRAN.R-project.org/package=TCGA2STAT ).

Article Snippet: CaSki and HeLa cells were also transfected with 2 μg of an E2F1 expression vector (pRcCMV-HA-E2F1; Addgene, Watertown, MA) with X-tremeGENE HP DNA transfection reagent (Roche), and total RNA from the cells with E2F1 overexpression was analyzed for RNASEH2A expression by real-time RT-qPCR.

Techniques: Expressing, RNA Expression, Western Blot, Quantitative RT-PCR, Plasmid Preparation