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: Plant Methods

Article Title: An optimised CRISPR Cas9 and Cas12a mutagenesis toolkit for Barley and Wheat

doi: 10.1186/s13007-024-01234-y

Figure Lengend Snippet: Performance of Cas9 and Cas12a components in barley and wheat. 2A . Percentage of T0 barley plants containing Sanger sequencing detected mutations in five target genes (chr7Hg0693091, chr5Hg0460461, chr2Hg0110011, chr5Hg0454731, chr4Hg0361381) using three different Cas9 CDS variants. HsCas9 = Human codon optimised CDS, AtCas9 + 1int = Arabidopsis codon optimised Cas9 with 1 intron, ZmCas9 + 13int = Zea mays codon optimised CDS with 13 introns. For the data in Fig. 2A, B, C and F, mutation frequencies were analysed by generalised linear modelling (GLM) of proportions of T0 plants which were mutagenised, using a binomial distribution with a logit link function. In Fig. 2A, in a model of Gene + Cas9 variant, differences between Cas9 variants were highly significant (chi-squared [Chi2] = 65.4, 2 degrees of freedom [df], P < 0.001) but variation between genes was not (Chi2 = 1.90, 4 df, P = 0.1). Error bars are standard errors of mean efficiency for each construct averaged over genes. 2B . Percentage of T0 barley plants containing Sanger sequencing detected mutations in three simultaneously targeted genes, using three different Cas9 guide architectures. Architecture A has all 12 guides driven by their own U3 or U6 promoter. Architecture B has a single rice ubiquitin promoter driving all 12 guides which are separated by hammerhead and hepatitis delta virus ribozymes. Architecture C has a single rice ubiquitin promoter driving all 12 guides which are separated by CSY4 cleavage sequences. Csy4 is co-expressed within this architecture. The statistical model was Gene + Architecture; the Architecture effect was very highly significant (Chi2 = 10.9, 2 df, P < 0.001) but the Gene effect was not (Chi2 = 1.06, 2 df, P = 0.3). Error bars standard errors of mean efficiency for each architecture averaged over genes. 2 C . Percentage of T0 wheat plants mutagenised in A, B & D subgenomes of target RLK genes using the Cas9 system that worked best in barley ( Zm Cas9 + 13int/Guide architecture A). Variation in mutation frequencies between genomes was not statistically significant (Chi2 = 1.05, 2 df, P = 0.4). 2D . Overall mutagenesis efficiencies of Cas12a CDS variants over three barley gene targets (chr6Hg0653951, chr7Hg0684671 and chr2Hg0138701). 20 T0 plants were screened for each CDS by PCR and Illumina sequencing at each of the three gene target sites. The percentage of mutant alleles detected for each plant in the three genes are plotted: Median indicated by horizontal bar, mean with an X, boxes contain the first and third quartiles, whiskers extend to a maximum 1.5 times the interquartile range beyond which outliers are marked with a dot. Data on frequencies of mutated alleles in Fig. 2D and E were analysed by analysis of variance (anova). In Fig. 2D, the model used was Gene * CDS (* is the crossing operator) and all three terms were highly significant (Gene: F = 140, 2 numerator df [ndf]; CDS: F = 151, 6 ndf; Gene.CDS: F = 15.7, 12 ndf; 399 denominator df [ddf] and P < 0.001 for all three terms). 2E . Comparison of the mutagenesis efficiencies of Cas12a V2 and V3 guide architectures at the recalcitrant barley target chr7Hg0684671. 20 T0 plants were screened for both V2 & V3 architectures by PCR and Illumina sequencing at the chr7Hg0684671 gene target site. The percentage of mutant alleles detected for each plant are plotted: Median indicated by horizontal bar, mean with an X, box contain the first and third quartiles, whiskers extend to a maximum 1.5 times the interquartile range beyond which outliers are marked with a dot. The statistical model was Gene*CDS and all three terms were highly significant (Gene: F = 78.9, 2 ndf; CDS: F = 90.6, 3 ndf; Gene.CDS: F = 34.6, 6 ndf; 228 ddf and P < 0.001 for all three terms). 2 F . Simultaneous targeting of two wheat genes ( Ta GW2 & Ta GW7) using Cas12a parts proven in barley. The mean percentage of T0 plants mutagenised across the two genes in all subgenomes is shown for the three constructs tested. ttHsCas12a = human codon optimised Cas12a CDS with D156R, ttAtCas12 + 8int = AtCas12a CDS with D156R & 8 introns. V2 = guides in V2 architecture array, V3 = guides in V3 architecture array. Variation between constructs was very highly significant (Chi2 = 68.4, 2 df, P < 0.001). Error bars are standard errors of mutagenesis frequencies for each construct averaged over genes and genomes. All statistical analysis was done with the package Genstat 23rd edition (VSN International, U.K.)

Article Snippet: Previously we have had good results with multiplex editing in Medicago truncatula [ ] using a CSY4 cleavage system.

Techniques: Sequencing, Mutagenesis, Variant Assay, Construct, Ubiquitin Proteomics, Virus, Illumina Sequencing, Comparison