Journal: Nature communications

Article Title: Delivery of Prime editing in human stem cells using pseudoviral NanoScribes particles.

doi: 10.1038/s41467-024-55604-0

Figure Lengend Snippet: Fig. 3 | Expression of pegRNA with a POL II-promoter enhances prime editing delivery. a Structure of post-splicing ipegRNA and epegRNA.Created in BioRender. Ohlmann, T. (2024) https://BioRender.com/g43z718. b Efficacy of PE measured in SWYS cells transduced with GAG-PEV4-VLPs produced from cells expressing intronic-pegRNAs. Production was performed with addition of the Csy4 protein or its non-cleaving mutant Csy4H29A. (n = 9) c Comparison of GAG-PEV4 and GAG-PE- V7 for the production of ipeg-loaded VLPs. (n = 9). d Impact of ipegRNA on VLP-

Article Snippet: Plasmids coding for PE2 (#132775), PEmax (#174820) and Csy4 (55196) were obtained from Addgene.

Techniques: Expressing, Transduction, Produced, Mutagenesis, Comparison

Journal: Nature communications

Article Title: Rational design of inducible CRISPR guide RNAs for de novo assembly of transcriptional programs.

doi: 10.1038/ncomms14633

Figure Lengend Snippet: Figure 2 | Design and optimization of protein-responsive iSBH-sgRNAs. (a) Conceptual framework underlying conditional spacer release using genetically encoded inducers (endoribonucleases). Grafting the Csy4 RNA motif onto the SBH stem allows OFF- to ON-state transition in the presence of the CRISPR-associated endoribonuclease Csy4. (b) Sequence and RNA secondary structure of the Csy4-responsive iSBH(0B)Csy4(full)CTS1 and corresponding control mutant variant iSBH(0B)Csy4m(full)CTS1 (base pair change (yellow) renders the recognition sequence insensitive to Csy4 cleavage). Red arrow indicates Csy4 cleavage site. (c) Representative flow cytometry scatter plots (EYFP reporter fluorescence against iBlue sgRNA transfection) reveal complete silencing in the absence of inducer (decoy ¼ empty plasmid). Robust reporter activation observed in the presence of Csy4 is lost when mutating Csy4-iSBH. (d–f) Optimization of Csy4-iSBH designs. RNA secondary structures (CTS1 spacer; red arrow Csy4 cleavage site) (d) and representative CRISPR-TR assay flow cytometry scatter plots ( þ Csy4 ON-state) (e) for iSBH(0B)Csy4 full, medium and nano stems. Quantification of EYFP activation score (see Methods) using the three iSBH variants in the presence of a decoy plasmid or Csy4 inducer from three biological replicates (n ¼ 3, mean±s.d.; a.u., arbitrary units) (f). Flow cytometry plot insets display % of activated cells (double iBlue þ ve and EYFP þ ve, green) and median reporter fluorescence intensity for this population (orange).

Article Snippet: The NLS sequence contained in the pX330 vector was PCR amplified (Fwd_pX330_NLS and Rev_pX330_NLS) and cloned between HindIII and NheI sites upstream of Pseudomonas aeruginosa Csy4 (human codon optimized) in the PGK1p-Csy4-pA vector (gift from Timothy Lu (Addgene plasmid 55196)).

Techniques: CRISPR, Sequencing, Control, Mutagenesis, Variant Assay, Cytometry, Transfection, Plasmid Preparation, Activation Assay, Flow Cytometry

Journal: Advanced Science

Article Title: Enhancing Prime Editing Efficiency Through Modulation of Methylation on the Newly Synthesized DNA Strand and Prolonged Expression

doi: 10.1002/advs.202417790

Figure Lengend Snippet: One‐step generation of multiple precise genome edits via the Csy4‐mediated processing system. A) Structure of the vectors of three‐pegRNAs containing cPE2 and epiPE2 and statistical analysis of the efficiencies of individual pegRNAs. B) Summary of mutation frequencies at three target sites by the three‐pegRNA epiPE2‐Csy4 system in HEK293T cells. C) Schematic representation of the genotyping results for all 48 cell colonies categorized as biallelic mutation, monoallelic mutation and wild type. D) Structure of the vector used to express pegRNAs 1 to 10 via the U6 promoter and efficiency analysis. E) Structure of the vector used to express pegRNAs 1 to 10 via the EF1α promoter and efficiency analysis.

Article Snippet: To produce the epiPE2‐Csy4 vector, the Csy4 expression cassette was synthesized by blocking the AarI site (Genscript) and fused to epiPE2.

Techniques: Mutagenesis, Plasmid Preparation