Journal: Scientific Reports

Article Title: Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli

doi: 10.1038/srep13477

Figure Lengend Snippet: ( A ) Sequence of the designed sgRNA template. sgRNA targets the non-template DNA strand of the gene-coding region. Base-pairing nucleotides (20 bp) are shown in orange. The dCas9-binding hairpin is in blue. The PAM sequence is shown in red. The Trc promoter is shown in grey. ( B ) This CRISPRi system consists of an inducible dCas9 protein and a designed sgRNA chimera. The dCas9 mutant gene contains two silencing mutations of the RuvC1 and HNH nuclease domains. The sgRNA chimera contains four functional domains: a Trc -inducible promoter, a 20-nucleotide (nt) complementary region for specific DNA binding, a 42-nt dCas9-binding hairpin and a 40-nt transcription terminator derived from S. pyogenes .

Article Snippet: A catalytically dead Cas9 mutant (dCas9) was codon-optimized and synthesized by GenScript (Nanjing, China) (see for the dCas9 sequence). pACYC-dCas9 was constructed by digesting dCas9 from pUC57-dCas9 (GenScript, Nanjing, China) into Nco I/ Hin dIII sites of pACYCDuet-1.

Techniques: Sequencing, Binding Assay, Mutagenesis, Functional Assay, Derivative Assay

Journal: Scientific Reports

Article Title: Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli

doi: 10.1038/srep13477

Figure Lengend Snippet: Control strains contained the (2S)-naringenin heterologous pathway without an RNA-guided dCas9:sgRNA system. The sgRNA-expressing plasmids repressing single or multiple genes were further transformed into the control strain to investigate the effects of these systems on (2S)-naringenin production. Final OD 600 values, average specific growth rates and concentrations of p -coumaric acid and (2S)-naringenin were measured from production strains after a total fermentation time of 48 h.

Article Snippet: A catalytically dead Cas9 mutant (dCas9) was codon-optimized and synthesized by GenScript (Nanjing, China) (see for the dCas9 sequence). pACYC-dCas9 was constructed by digesting dCas9 from pUC57-dCas9 (GenScript, Nanjing, China) into Nco I/ Hin dIII sites of pACYCDuet-1.

Techniques: Control, Expressing, Transformation Assay

Journal: Scientific Reports

Article Title: Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli

doi: 10.1038/srep13477

Figure Lengend Snippet: Plasmids used for multiple gene perturbations.

Article Snippet: A catalytically dead Cas9 mutant (dCas9) was codon-optimized and synthesized by GenScript (Nanjing, China) (see for the dCas9 sequence). pACYC-dCas9 was constructed by digesting dCas9 from pUC57-dCas9 (GenScript, Nanjing, China) into Nco I/ Hin dIII sites of pACYCDuet-1.

Techniques:

Journal: Cell Death Discovery

Article Title: The Neurod1/4-Ntrk3-Src pathway regulates gonadotrope cell adhesion and motility

doi: 10.1038/s41420-023-01615-7

Figure Lengend Snippet: A Heatmap of Neurod/g gene expression in αT1-1, αT3-1, and LβT2 cells according to published RNA-seq (GSE104513). Color scale represents the normalized expression values. B Expression of Neurod1 and Neurod4 in αT1-1, αT3-1, and LβT2 cells. Gene expression levels were measured by RT-qPCR and normalized to Ppia (Cyclophilin A). Data are the normalized mean ± SEM of four independent experiments. Neurod1 and Neurod4 transcript levels were compared between the three cell lines by ANOVA followed by Tukey multiple comparison test. *** p < 0.001. C Chromatin accessibility of Neurod1 and Neurod4 loci in αT1-1, αT3-1, and LβT2 cells. Chromatin accessibility was investigated by ATAC-seq (assay for transposase-accessible chromatin with high-throughput sequencing) in the three gonadotrope cell lines. ATAC-seq tracks are shown for Neurod1 (left part of the panel) and Neurod4 (right part of the panel) loci. Gene exons are depicted as black boxes and proximal promoters are depicted as gray box in 5 prime of the ORFs. For each cell line, identified accessible chromatin regions are depicted as colored rectangles under each track. D Cis -regulatory activity of Neurod4 potential enhancers in gonadotrope cells. αT1-1, αT3-1, and LβT2 cells were transiently transfected with a GL4 luciferase reporter cassette under the control of Neurod4 promoter, here referred as R2 (pR2-GL4) or with this construct harboring potential cis-regulatory regions cloned in 3 prime of the luciferase cassette (pR2-GL4-Rx constructs, with x = R1 to R6 except R2). The activity of pR2-GL4 was normalized to the control pGL4 empty backbone activity (inset) and then pR2-GL4-Rx activities were normalized to pR2-GL4 activity. Relative luciferase activities are the normalized ratio between firefly luciferase over renilla luciferase. ANOVA followed by Dunnett’s multiple comparison test was performed independently for each cell line. Results are the mean ± SEM of six independent experiments. Significant difference with pR2-GL4 construct: ** p < 0.01; *** p < 0.001. E Deletion of the genomic sequence of Neurod4 R6 enhancer and Neurod1 ORF was carried out in αT3-1 cells using CRISPR/Cas9. For Neurod4 , genotyping PCR amplified a 1100 bp amplicon in WT cells while stable Neurod4 mutant cells (ΔND4) carried a deletion of about 320 bp, removing the R6 enhancer (left part of the panel, 780 bp amplicon). For Neurod1 , a 780 bp amplicon was detected in WT cells while stable Neurod1 mutant cells (ΔND1) carried a deletion of about 500 bp, which encompassed Neurod1 ATG first codon (right part of the panel, 280 bp amplicon). The double mutant αT3-1 cell line (ΔΔ) was generated by deleting R6 enhancer on the Neurod1 mutant background. F For each genotype, homozygous clones were tested for Neurod4 and Neurod1 expression by RT-qPCR. Gene expression levels were normalized to Ppia . Data are the normalized mean ± SEM of four independent experiments. Gene expression levels of mutant clones were compared to WT by ANOVA followed by Dunnett’s multiple comparison test. **** p < 0.0001.

Article Snippet: For Ptpn13 knockdown, cells were co-transfected with a pLV hUbC-Zim3KRAB hdCas9-T2A-GFP, expressing a dead Cas9 (Addgene #53191) fused with the repressor KRAB domain of the human ZIM3 gene (as described in [ ]), and gRNA targeting Ptpn13 proximal promoter described in [ ] and supplementary table ).

Techniques: Gene Expression, RNA Sequencing, Expressing, Quantitative RT-PCR, Comparison, Next-Generation Sequencing, Activity Assay, Transfection, Luciferase, Control, Construct, Clone Assay, Sequencing, CRISPR, Amplification, Mutagenesis, Generated

Journal: Nature Communications

Article Title: Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences

doi: 10.1038/s41467-022-29202-x

Figure Lengend Snippet: a Chemical structures of universal and degenerate bases used in this study. b List of DNA targets corresponding to sequences in the ABO gene based on clinical polymorphism data. SNPs are indicated with red lettering. Allele frequency indicates either the current tallied allele frequency or the statistically predicted frequency (for sequences containing multiple SNPs). The PAM sequence is underlined. Bar graphs showing the relative amount of DNA cleavage resulting from in vitro reactions containing Cas9 with ( c ) double or ( d ) triple modified-crRNAs and the variant DNA target sequences indicated. Locations of the universal bases in the crRNA sequence are indicated with red [*]. rI = Ribose Inosine, dI = Deoxyribose Inosine, mI = 2′-O-Methyl ribose Inosine, dN = Deoxyribose 5′-Nitroindole, dK = Deoxyribose K, dP = Deoxyribose P; Mean with individual data points shown ( n = 2 independent experiments). Bar graphs showing the relative amount of DNA cleavage resulting from in vitro reactions containing Cas9 with ABO-RNA or ABO-rI-2 versus derivatives of the ( e ) ABO-T5 or ( f ) ABO-T7 target sequences. Base combinations listed along the x -axis correspond to the positions indicated by a red ‘X’ in the reference sequence. The PAM sequence is underlined; Mean with individual data points shown ( n = 2 independent experiments). Reactions were performed using fixed concentrations of gRNA (80 nM) and Cas9 (40 nM). Cleavage percentages were calculated from corresponding agarose gels using densitometry software (ImageJ).

Article Snippet: Briefly, E. coli Rosetta (DE3) cells were transformed with a plasmid encoding either S. pyogenes Cas9 or catalytically-dead S. pyogenes Cas9 (dCas9) fused to an N-terminal 6xHis-tag, MBP, and TEV site (Addgene #39312 and #39318, respectively).

Techniques: Sequencing, In Vitro, Modification, Variant Assay, Software

Journal: Nature Communications

Article Title: Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences

doi: 10.1038/s41467-022-29202-x

Figure Lengend Snippet: a List of ABO variant DNA target sequences (ABO-T1-T8) assayed in cells. Positions of SNPs are indicated with red lettering. The PAM sequence is underlined. b Bar graphs showing the relative amount of DNA cleavage resulting from in vitro reactions containing Cas9 with ABO-RNA or ABO-rI-2 versus the indicated DNA target sequences. Assays were performed using fixed concentrations of gRNA (80 nM) and Cas9 (40 nM); Mean with individual data points shown ( n = 2 independent experiments). c Schematic outlining the framework for the fluorescence-based assay used to evaluate cleavage of the ABO variant target sequences in cells. d Representative FACS plot showing the distribution of RFP and GFP positive cells. Dual positive cells appear in the top right quadrant. e Table showing normalized %GFP + /all %RFP + events corresponding to cleavage of the indicated target sequences in cells by Cas9 using either ABO-RNA or ABO-rI-2; Mean ± S.D. shown ( n = 3 independent samples).

Article Snippet: Briefly, E. coli Rosetta (DE3) cells were transformed with a plasmid encoding either S. pyogenes Cas9 or catalytically-dead S. pyogenes Cas9 (dCas9) fused to an N-terminal 6xHis-tag, MBP, and TEV site (Addgene #39312 and #39318, respectively).

Techniques: Variant Assay, Sequencing, In Vitro, Fluorescence

Journal: Nature Communications

Article Title: Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences

doi: 10.1038/s41467-022-29202-x

Figure Lengend Snippet: a List of DNA target sequences derived from the HIV-1 protease gene containing evolved SNPs detected in patient samples. SNPs position(s) are indicated with red lettering. The PAM sequence is underlined. b Bar graphs showing the relative amount of DNA cleavage resulting from in vitro reactions containing Cas9 with HIV-RNA or HIV-rI-1 versus the indicated DNA target sequences. A scrambled crRNA with the sequence 5′-AUUCUUGCUCUGCUCUCUUCGUC-′3 was used as a negative control. Assays were performed using fixed concentrations of crRNA (125 nM) and Cas12a (100 nM); Mean with individual data points shown ( n = 2 independent experiments). c Representative gels of the in vitro cleavage assay results for Cas12a with HIV-RNA or HIV-rI-1 versus the indicated DNA target sequences. The bottom two bands in the gel represent the cleaved DNA substrate while the top band corresponds to the undigested substrate. Cleavage experiments were performed in duplicate with similar results. d Diagram outlining the DETECTR assay. e Bar graph indicating the fluorescence signal obtained in the DETECTR assay using Cas12a in combination with either HIV-RNA or HIV-rI-1 and samples containing the indicated target sequences. Max fluorescence values were normalized to background; Mean with individual data points shown ( n = 3 independent experiments).

Article Snippet: Briefly, E. coli Rosetta (DE3) cells were transformed with a plasmid encoding either S. pyogenes Cas9 or catalytically-dead S. pyogenes Cas9 (dCas9) fused to an N-terminal 6xHis-tag, MBP, and TEV site (Addgene #39312 and #39318, respectively).

Techniques: Derivative Assay, Sequencing, In Vitro, Negative Control, Cleavage Assay, Fluorescence