Journal: Virulence

Article Title: The stress of carrying CRISPR-Cas

doi: 10.1080/21505594.2025.2541701

Figure Lengend Snippet: Expression and functionality of CRISPR-Cas system in S. anginosus . (a) RT-PCR performed with 20 ng isolated RNA of S. anginosus SK52 (WT), cas9 deletion (∆ cas9 ), cas9 complementation (∆ cas9:cas9 ) and CRISPR array deletion (∆CRISPR) strain verifying expression of cas9 using primer 13/41. Complete DNase digestion was controlled by conducting PCR without RT step. Controls with genomic DNA of S. anginosus SK52 are indicated by “+” and negative controls without nucleic acid by “-“. The 1 kb plus DNA ladder (Invitrogen) served as molecular weight marker. (b) Confirming the functionality of S. anginosus CRISPR-Cas system by interference with plasmid transformation. Relative transformation efficiencies of created plasmids normalized to the empty vector pAT28 were obtained via competence-based transformation assay. Vectors were designed to contain an endogenous spacer of S. anginosus SK52 CRISPR-Cas system followed by a functional or non-functional PAM sequence, resulting in the CRISPR-targeted plasmid pAT28_SP1_TGG and the non-targeted plasmid pAT28_SP1_AAA. Mean values and standard deviations of six independent experiments are shown. Mann-Whitney-U test was performed to illustrate significant differences as indicated (** p < 0.01).

Article Snippet: Inserts were constructed by annealing oligonucleotides 31/32 and 33/34 and were subsequently cloned into the plasmid backbone pAT28 (both digested with Eco RI and Bam HI [New England Biolabs]).

Techniques: Expressing, CRISPR, Reverse Transcription Polymerase Chain Reaction, Isolation, Molecular Weight, Marker, Plasmid Preparation, Transformation Assay, Functional Assay, Sequencing, MANN-WHITNEY

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