Journal: Nature

Article Title: Developmental dynamics of two bipotent thymic epithelial progenitor types

doi: 10.1038/s41586-022-04752-8

Figure Lengend Snippet: a , Schematic of the CRISPR–Cas9-based barcoding system. DSB, double-strand break. b , Location of the target site in exon 3 of the mouse Hprt gene. c , Examples of barcodes; the germline sequence is shown with the sgRNA target and protospacer adjacent motif (PAM) sequences indicated at top. Nucleotide additions and deletions (dashes) are indicated in red. d , Frequencies of individual barcodes in decreasing order. e , Number of Foxn1 -expressing TECs in the thymic rudiment of E12.5 embryos (left; n = 5) and numbers of different barcodes in the thymi of mice of different ages (right): E16.5, n = 6; P0, n = 5; P12–P15, n = 11; >P16, n = 12. The dotted lines indicate the range of the numbers of progenitors previously inferred from medullary islet counts in adult mice . f , Enrichment of shared barcodes in the Ly51 – UEA-1 + mTEC and Ly51 + UEA-1 – cTEC fractions of mice of different ages. Enrichment values were significantly different in the comparison of mice at P0 and >3 weeks (w) ( P = 0.009, one-sided Wilcoxon test). E16.5, n = 6; P0, n = 5; ~2 weeks, n = 11; >3 weeks, n = 11. For e and f , boxes extend from the 25th to 75th percentile; whiskers extend to the largest and smallest values; and the median is indicated. See the for a definition of the enrichment value E m . g , Co-occurrence probability of rare barcodes across pairs of samples highlighting enhanced co-occurrence in mTEC (m) and cTEC (c) fractions of the same mouse; individual mice are identified by number. Data are shown for n = 18 mice. h – k , P values (–log 10 ) of barcode frequencies indicating co-occurrence of individual barcodes in progenitor and mature TEC fractions (as defined in Fig. ) at different time points. For g – k , P values were calculated as described in the and corrected for multiple testing by the Benjamini–Hochberg method. The red numbers refer to clones discussed in the text.

Article Snippet: The hU6-sgRNA Hprt transgene was cloned as a NotI fragment into the Bluescript vector and consists of the human U6 promotor (nucleotides 1–264 in GenBank accession number JN255693 ) followed by the mouse Hprt target sequence (5′-GATGGGAGGCCATCACATTGG-3′; nucleotides 255–274 in GenBank accession number J00423 ), the sgRNA backbone (nucleotides 218–139 (reverse complement) in Addgene plasmid 42250) and a short 3′ sequence (TTTTTTGGAA); for injection into fertilized eggs, the construct was linearized with SacI.

Techniques: CRISPR, Sequencing, Expressing, Comparison, Clone Assay

Journal: Nature

Article Title: Developmental dynamics of two bipotent thymic epithelial progenitor types

doi: 10.1038/s41586-022-04752-8

Figure Lengend Snippet: a , Schematic of the components of the hU6:sgRNA Hprt transgene; key features are indicated by name and are colour-coded; the bar representing the hU6 promotor sequence was truncated. b , Nucleotide sequence of the hU6:sgRNA Hprt transgene construct (colour code as in a ). c – e , Frequencies of individual barcodes in decreasing order from left to right grouped by the degree of occurrence in the cohort of mice analysed here (n = 33); colours indicate those barcodes that satisfy the criterion indicated at the top right of each plot. f , Scatter plots of barcode frequencies for mTECs and cTECs from the same mouse versus barcode frequencies in cTECs isolated from two different mice. g , Fraction of informative barcodes observed in the TEC compartment of individual mice; informative barcodes are those whose P values indicate a significant deviation ( P b,i < 0.001 for barcode b in sample i ) from the barcode frequencies expected from the background model. Since these values represent singular data points, statistical comparisons were not done. Barcode data are listed in Supplementary Tables –

Article Snippet: The hU6-sgRNA Hprt transgene was cloned as a NotI fragment into the Bluescript vector and consists of the human U6 promotor (nucleotides 1–264 in GenBank accession number JN255693 ) followed by the mouse Hprt target sequence (5′-GATGGGAGGCCATCACATTGG-3′; nucleotides 255–274 in GenBank accession number J00423 ), the sgRNA backbone (nucleotides 218–139 (reverse complement) in Addgene plasmid 42250) and a short 3′ sequence (TTTTTTGGAA); for injection into fertilized eggs, the construct was linearized with SacI.

Techniques: Sequencing, Construct, Isolation

Journal: Cell Reports Medicine

Article Title: Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy

doi: 10.1016/j.xcrm.2021.100374

Figure Lengend Snippet:

Article Snippet: DNA constructs for retro-/lentiviral transduction had the following structure: Human Kozac sequence followed by TCR β (including as indicated either human TRBC or murine TRBC with additional cysteine bridge , , ), followed by P2A, followed by TCR α (including as indicated either human TRAC or murine TRAC with additional cysteine bridge , , ), cloned into the pMP71 vector (kindly provided by Wolfgang Uckert, Berlin, Addgene plasmid backbone #108214) or into the pCDH-EF1 vector (Addgene plasmid #72266).

Techniques: Recombinant, CRISPR, Electroporation, Amplification, Software

Journal: bioRxiv

Article Title: Phase separation of mycobacterial Rho factor is associated with acid stress

doi: 10.1101/2025.08.28.672908

Figure Lengend Snippet: (A) Wild-type strain harbouring the CRISPRi system with different sgRNAs for the promoter region of rho and three positive controls ( DNAβ, MSMEG_0317 , and rho ). (B) Wild-type strain containing the CRISPRi plasmid and a second copy of rho . Strains with pJR962 derivatives expressing sgRNAs were grown to the stationary phase in TSBT in the absence of aTc. The cells were serially diluted 10-fold, and 5 µL of each dilution was spotted onto TSA without (left) or with (right) aTc, which induces expression of the CRISPRi system. Positive controls were designed by .

Article Snippet: The sgRNA oligos were cloned into the BsmBI-digested CRISPRi backbone (plJR962 - Addgene plasmid #115162).

Techniques: Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Phase separation of mycobacterial Rho factor is associated with acid stress

doi: 10.1101/2025.08.28.672908

Figure Lengend Snippet: Localization patterns for Msm Rho and its derivatives after 4-hour exposure to TSBT or pH 4 (citrate/phosphate buffer): (A) Wild-type Rho; (B) ΔIDR; (C) Δinitial; (D) Δinsertion. All cultures were supplemented with aTc to repress the native copy of rho through the CRISPRi system. Demographs on the right display the normalized Dendra intensity as heatmaps along the medial axis of single cells (n = 75) which are sorted by length. All analyses were carried out from three independent experiments, and representative images are shown. All scale bars, 2 µm.

Article Snippet: The sgRNA oligos were cloned into the BsmBI-digested CRISPRi backbone (plJR962 - Addgene plasmid #115162).

Techniques: