Journal: Biotechnology and bioengineering

Article Title: Domesticating a food spoilage yeast into an organic acid-tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii.

doi: 10.1002/bit.27576

Figure Lengend Snippet: FIGURE 3 Zygosaccharomyces bailii ZbADE2 disruption experiment overview. Transfer RNA based maturation of gRNA (a). Design of ZbADE2 gRNA and donor DNA (b). Screening of ZbADE2 mutants (c), the mutants show distinctive red color accumulation in adenine‐deficient plate. Sequencing of ZbADE2 (d), the results show CRISPR Cas9 mediated mutation in ZbADE2 gene. gRNA, guide RNA [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The original plasmid pUDP004 containing gRNA and Cas9 expression cassettes were obtained from ADDGENE (catalog #101165).

Techniques: Disruption, Sequencing, CRISPR, Mutagenesis

Journal: Frontiers in Genome Editing

Article Title: Cytosine base editors optimized for genome editing in potato protoplasts

doi: 10.3389/fgeed.2023.1247702

Figure Lengend Snippet: C-to-T conversion derived from cytosine base editors A3A, rA1, and CDA1 optimized for potato. (A) Three cytosine base editor constructs comprising a cassette driving expression of the gRNA (target conferring part, GGT​C 4 C 5 TTG​GAG​C 12 AAA​AC 17 TGG) from the St U6-1 promotor , the Sp Cas9 nickase (nCas9) in fusion with one of the deaminases hAPOBEC3A (A3A) from human, evo_rAPOBEC1 (rA1) from rat, or sea lamprey evo_ Pm CDA1 (CDA1), followed by a uracil-DNA glycosylase inhibitor (UGI), interspaced by long flexible linkers are depicted. NLS, nuclear localization signal; serine (S) and glycine (G) linkers: SG and SGGS. (B) Chromatograms from direct sequencing of PCR products from the protoplast cell pool transformed with the base editing constructs with A3A showing 39% (C4), 43% (C5), and 36% (C12) editing (sample A3A replicate 4), rA1 showing 29% (C4) and 28% (C5) (sample rA1 replicate 3), and CDA1 showing 38% (C4), 38% (C5), and 21% (C12) (sample CDA1 replicate 2) C-to-T conversion in the target region (exon 1 of GBSS1 (cultivar Desiree)) when compared to the WT sequence. Target Cs in the gRNA and adjacent PAM site are shown in green and red, respectively. (C) C-to-T conversion (%) of Cs within (C4, C5, C12, and C17) and most closely adjacent (−C19, −C8 and C25) to the gRNA of protoplasts transformed with A3A, rA1, and CDA1 as evidenced by EditR analysis. Numbers 1–4 indicate replicates. (D) Average percentage of reads with C-to-T conversion (%) rates of protoplasts transformed with A3A, rA1, and CDA1 as shown for Cs within (C4, C5, C12, and C17) and most closely adjacent (−C19, −C8 and C25) to the gRNA. Data are shown as mean ± sd of four biological replicates.

Article Snippet: The basic construct, Sp Cas9/ St U6-1::sgRNA1, comprising the 35SPPDK:: Sp Cas9 cassette, driving the expression of the codon-optimized Streptococcus pyogenes Cas9 nuclease ( Sp Cas9) originally from the plasmid pHBT-pcoCas9 ( ) (Addgene plasmid #52254) and the St U6-1 promoter::sgRNA-1 cassette ( St U6-1 promoter (NCBI accession no. Z17290)) described in the work of ) was used as the basis for generation of the base editing constructs.

Techniques: Derivative Assay, Construct, Expressing, Sequencing, Transformation Assay

Journal: Science signaling

Article Title: A small sustained increase in NOD1 abundance promotes ligand-independent inflammatory and oncogene transcriptional responses

doi: 10.1126/scisignal.aba3244

Figure Lengend Snippet: (A-C) qPCR (A), immunoblot (B) and flow cytometry plots (C) showing oncogene expression in THP-1 NOD1 cells in absence and presence of DOX. (D) Immunoblot showing the abundance of the oncogene C-MYC and the phosphorylation (p) of AKT in THP-1 NOD1 cells in presence or absence of DOX. (E) Kinetics of C-JUN and C-MYC expression following ligand treatment as measured by qPCR. Grey arrows indicate times of recurring ligand addition (0, 24 and 48 h). Expression at each time point is represented relative to the untreated control at that timepoint (dashed line set to 1). (F-H) qPCR (F) and immunoblots (G, H) for the indicated genes in THP-1 NOD1 cells following CRISPR/Cas9 mediated ablation of FLAG-NOD1 in three independent single cell clones (NF1, NF2 and NF3). (I) qPCR for the indicated genes in THP-1 NOD1 cells following CRISPR/Cas9 mediated ablation of RIPK2 in three independent single cell clones. NT: non-targeting gRNA. NF: gRNA targeting FLAG-NOD1. RIPK2: gRNA targeting RIPK2. Data in (A) to (I) are representative of three independent experiments. Error bars on graphs are mean±SEM of at least three biological replicates and where not visible in (E) are shorter than the height of the symbol. p values from an unpaired t test (two-tailed) without assuming a consistent standard deviation are shown. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: For CRISPR/Cas9 targeting of RIPK2 or FLAG NOD1 , the guide RNA targeting RIPK2 or FLAG NOD1 (IDT) was cloned into a single self-inactivating lentivirus plasmid pRRL-U6-empty-gRNA-MND-Cas9-t2A-Puro, that expresses a Cas9-T2A-puromycin resistance cassette controlled by an MND promoter ( 45 ).

Techniques: Western Blot, Flow Cytometry, Expressing, CRISPR, Clone Assay, Two Tailed Test, Standard Deviation

Journal: Science signaling

Article Title: A small sustained increase in NOD1 abundance promotes ligand-independent inflammatory and oncogene transcriptional responses

doi: 10.1126/scisignal.aba3244

Figure Lengend Snippet: (A) Histograms (left) and quantification (right) of NOD1 protein in THP-1 cells treated with scramble or miR-191 LNA or miR-15b/16 TSB. (B) qPCR for the indicated oncogenes in cells treated with miR-15b/16 TSB. Gene expression at each timepoint is represented relative to the scramble LNA-treated control at that timepoint (dashed line set to 1). (C) qPCR for miR-15b, miR-16, NOD1 and oncogenes in ex vivo human monocytes following inhibition of miR-15b and miR-16 with LNA. Gene expression is represented relative to that in the scramble LNA control (dashed line set to 1). (D) qPCR for miR-15b, miR-16 and NOD1 (left) and the indicated oncogenes (right) in THP-1 cells following CRISPR/Cas9 mediated reduction of miR15b/16 in two independent clones analyzed 4–6 weeks after miR-15b/16 targeting and single cell cloning. Gene expression is represented relative to that in the non-targeting (NT) gRNA control (dashed line set to 1). (E) qPCR for the indicated inflammatory genes in THP-1 cells following CRISPR/Cas9 reduction of miR15b/16. Data in A, B and D are representative of three independent experiments, and in E are representative of two independent experiments with three biological replicates per condition. In C, one representative of two independent donors is shown with three replicates per condition. Error bars on graphs are mean±SEM and p values from an unpaired t test (two-tailed) without assuming a consistent standard deviation are shown. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns: not significant.

Article Snippet: For CRISPR/Cas9 targeting of RIPK2 or FLAG NOD1 , the guide RNA targeting RIPK2 or FLAG NOD1 (IDT) was cloned into a single self-inactivating lentivirus plasmid pRRL-U6-empty-gRNA-MND-Cas9-t2A-Puro, that expresses a Cas9-T2A-puromycin resistance cassette controlled by an MND promoter ( 45 ).

Techniques: Expressing, Ex Vivo, Inhibition, CRISPR, Clone Assay, Two Tailed Test, Standard Deviation