Journal: Cell

Article Title: Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

doi: 10.1016/j.cell.2021.09.018

Figure Lengend Snippet: Design and results of Repair-seq screens for substitution prime editing outcomes, related to Figure 1 (A) Presumed model by which the reverse-transcribed 3′ DNA flap is permanently incorporated into the genome during prime editing ( Anzalone et al., 2019 ). (B) Installation of a G⋅C-to-C⋅G edit within a lentivirally integrated HBB sequence using SaPE2 and Sa-pegRNAs in HEK293T cells. PBS, primer binding site. Data represent the mean of n = 3 independent replicates. (C) Design of the prime editing Repair-seq lentiviral vector (pPC1000, additional details and full sequence information in ). In Repair-seq screens, a 453-bp region containing CRISPRi sgRNA sequence and prime editing outcome is amplified from genomic DNA for paired-end Illumina sequencing. The CRISPRi sgRNA is sequenced with a 44-nt Illumina forward read (R1), and the prime edited site (including +50 and –50 nick sites) is sequenced with a 263-nt Illumina reverse read (R2). Black triangles indicate positions of SaPE2-induced nicks programmed by Sa-pegRNA and Sa-sgRNAs. Sizes of all vector components are to scale. (D) Schematic of PE2, PE3+50, and PE3–50 prime editing configurations with SaPE2 protein (SaCas9 N580A fused to an engineered MMLV RT). (E) Validation of intended G⋅C-to-C⋅G editing at the lentivirally integrated Repair-seq edit site in HeLa cells expressing dCas9–BFP–KRAB cells. Bars represent the mean of n = 2 independent replicates. (F) Prime editing at the Repair-seq edit site with blasticidin selection in HeLa cells expressing dCas9–BFP–KRAB. SaPE2–P2A–BlastR prime editor was used for all conditions. Bars represent the mean of n = 2 independent replicates. (G) Functional annotation classes of the genes targeted by the pooled CRISPRi sgRNA library used in Repair-seq screens. (H–K) Knockdown of MSH2 , MSH6 , MLH1 , and PMS2 increases the frequency of the intended +6 G⋅C-to-C⋅G prime edit in all Repair-seq screens. Dots represent individual CRISPRi sgRNAs.

Article Snippet: Cells were washed once with PBS (Thermo Fisher Scientific) and lysed with gDNA lysis buffer (10 mM Tris-HCl, pH 8.0; 0.05% SDS; 800 units μL -1 proteinase K (New England BioLabs)) at 37°C for 1.5–2 h, followed by enzyme inactivation at 80°C for 30 min.

Techniques: Sequencing, Binding Assay, Plasmid Preparation, Amplification, Expressing, Selection, Functional Assay

Journal: Communications Biology

Article Title: Identifying the lungs as a susceptible site for allele-specific regulatory changes associated with type 1 diabetes risk

doi: 10.1038/s42003-021-02594-0

Figure Lengend Snippet: The locus tagged by rs6679677 was cloned within the 3′ UTR of a luciferase gene driven by a minimal promoter and transiently transfected into A549 and HepG2 cells. The relative enhancer activity for the ref and alt versions of the rs6679677 locus was calculated compared to the empty control vector (pMRAdonor2). Relative luminescence units (RLU) for the luciferase assay were normalised using the absorbance values for the beta-galactosidase assay. Results were plotted as the percentage of the ref_alleles enhancer activity (mean ± SD). Transfection experiments were repeated three times across n = 3 or n = 9 technical replicates in HepG2 (liver) and A549 (lung) cells, respectively. Representative results are shown for one transient transfection of A549 and HepG2 cells. Two-sided t -test was used for independent mean evaluation.

Article Snippet: On the day of transfection (24 following cell plating), ~75% confluent wells were co-transfected with luciferase plasmid DNA (i.e. 800 ng of pMPRA1:luc_rs6679677_ref, pMPRA1:luc_rs6679677_alt, or pMPRAdonor2 luciferase control) and a beta-galactosidase control plasmid (200 ng) using lipofectamine 3000 (ThermoFisher; according to the manufacturer’s instructions).

Techniques: Clone Assay, Luciferase, Transfection, Activity Assay, Control, Plasmid Preparation, β-Gal Assay

Journal:

Article Title: Requirement for an Intact T-Cell Actin and Tubulin Cytoskeleton for Efficient Assembly and Spread of Human Immunodeficiency Virus Type 1 ▿

doi: 10.1128/JVI.01469-06

Figure Lengend Snippet: Cytoskeletal inhibitors reduce cell-cell spread, as measured by qPCR. Equal numbers of JurkatLAI cells and uninfected target cells were mixed and incubated for 1, 3, and 6 h prior to cell lysis and purification of total cellular DNA. qPCR using pol primers was performed to measure de novo viral DNA synthesis. Data were normalized to HSA and are shown as ratios of HIV-1 DNA to HSA DNA. The data obtained with A2.01 target cells were used as baseline HIV-1 DNA synthesis at the different time points and were subtracted from the signal with A3.01 target cells. (A) JurkatLAI cells were either left untreated or incubated with Q4120 or AMD3100 prior to being mixed with A3.01 target cells to inhibit Env-CD4 or Env-CXCR4 interactions, respectively. (B) JurkatLAI cells were either left untreated or incubated with an inhibitor of actin remodeling (latrunculin, cytochalasin D, or jasplakinolide) prior to being washed and mixed with A3.01 cells. (C) JurkatLAI cells were either left untreated or incubated with an inhibitor of microtubule remodeling (colchicine or nocodazole). Bars represent the means for triplicate samples plus 1 SD.

Article Snippet: The CD4 + CXCR4 + Jurkat CE6.1 T-cell line (American Type Culture Collection) and the A3.01 T-cell line (Center for AIDS Reagents [CFAR], United Kingdom) were maintained in suspension cell growth medium (RPMI 1640 [Gibco BRL] supplemented with streptomycin [100 μg/ml], penicillin [100 U/ml], and 10% fetal calf serum [FCS]).

Techniques: Incubation, Lysis, Purification, DNA Synthesis