Journal: International journal of molecular sciences

Article Title: A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.

doi: 10.3390/ijms24076745

Figure Lengend Snippet: Figure 1. Overview of the design and dual-functionality of the TRE-Lox system. (a) Overall structure of the 5′ end of the murine cathepsin D (CatD) gene (CTSD) and its promoter region (PCTSD, dark gray), indicating the relative position of the two gRNAs (black arrows) used for CRISPR/Cas9- assisted homologous recombination. Note the placement of the TATA box (TATA) very close to the main transcription start site (TSS) (right-angle arrow), the presence of the initiation codon (ATG, dashed white line) within Exon 1 (Ex 1, light gray), and the presence of a splice donor (SD) and splice acceptor (SA) flanking Intron 1 (black line). (b) Structure of the TRE-Lox knock-in (KI) insert, illustrating the relative positions of the two tet-operons (tetO2, green) and one LoxP site (LoxP, light blue) within the 5′ untranslated region (5′UTR) and, within Intron 1, a tetracycline response element (TRE) comprised of seven tetO repeats (tetO7) and the second LoxP site. The relative placement of the puromycin resistance cassette (Puror, purple) flanked by two FRT sites (FRT, dark blue), which is excisable by Flp recombinase, is depicted using a curly bracket. (c) Downregulation of CTSD via the action of rtTRKRAB acting on the TRE-Lox insert. In the presence of Dox (red triangles), rtTRKRAB binds to the tetO repeats within both the 5′UTR and Intron 1, triggering methylation of histones in a radius of 2–3 kb, thereby remodeling the chromatin and silencing the CTSD gene. (d) Genetic deletion of CTSD via the action of Cre recombinase on the TRE-Lox insert. The figure depicts the end result of Cre-mediated recombination of the TRE-Lox KI insert, which causes removal of the initiation codon, the first portion of the coding region of Exon 1 encoding the signal peptide of CatD, and the 5′ end of Intron 1.

Article Snippet: Relevant regions (and their sources) were as follows: the 3′ end of Exon 1 and the 5′ portion of Intron 1 of murine CTSD (from C57Bl6/J mouse tail DNA); tetO2 (from Addgene plasmid #113892 [41]); TRE 3G and, separately, an FRT-flanked puromycin resistance cassette (both from Addgene plasmid #156430 [42]).

Techniques: CRISPR, Homologous Recombination, Knock-In, Methylation

Journal: bioRxiv

Article Title: The FANCM-RMI1/2 complex promotes genomic instability and PARP inhibitor sensitivity in BRCA2-deficicient cells

doi: 10.64898/2026.01.15.699681

Figure Lengend Snippet: a , Targeted homozygous insertion of CAS9 and OsTIR1 F74G at the TIGRE locus (left) results in constitutive CAS9 and TIR1 protein expression as assessed by western blot (right). b , Schematic representation of homozygous knockin of GFP-AID at (and in frame with) the start codon (N-terminus) of endogenous Brca1 or Brca2 , and PCR-genotyping of the resulting BRCA1- and BRCA2-degron cell lines. c , Venn diagram representation of total synthetic lethal and viable interactions scored in the BRCA1- and BRCA2-screens (FDR<0.05 and RRA<0.05). d , Top 10 Reactome pathways enriched among total BRCA1- and BRCA2- screen hits, ranked by signal (FDR-adjusted p-values with Benjamini-Hochberg correction). e , Dotplot representation of genes targeted by the genome-wide gRNA library ranked according to their synthetic viability (top) or synthetic lethality (bottom) scores across three different timepoints in the BRCA1-degron and f , BRCA2-degron screens. Statistically significant synthetic viable and lethal genes are shown as blue and red dots, respectively. A few top genes are labeled.

Article Snippet: The plasmid to simultaneously target CAS9 and OsTIR1(F74G variant) at the TIGRE locus (pFD155) was engineered by cloning Cas9-t2a-hygro and OsTir1(F74G)-V5 cassettes under pgk and CAG promoters respectively, into a plasmid containing 5’ and 3’ homology arms for targeted insertion at TIGRE (Addgene plasmid 92141).

Techniques: Expressing, Western Blot, Knock-In, Genome Wide, Labeling

Journal: bioRxiv

Article Title: Transcriptional perturbation of LINE-1 elements reveals their cis -regulatory potential

doi: 10.1101/2024.02.20.581275

Figure Lengend Snippet: A) Structure of the DOX-inducible LKF/AKF transgenes transfected in PGK-G10 cells for knock-in at Rosa26 . B) Following puromycin selection, colony picking and genotyping, 9 clones were analysed by qPCR for expression of the L1MdTf/L1MdA families using primers specific for the 5’UTR region, upon 48h of DOX treatment. C) Structure of the DOX-inducible dCas9-VPR transgene transfected in PGK-G10 cells for knock-in at Tigre . D) Following puromycin and blasticidin selections, colony picking and genotyping, 7 clones, including the control VPR-B7, were analysed for expression of L1MdTf/L1MdA upon DOX treatment (48h) by qPCR. Scissors indicate the approximate positions of gRNAs. TRE3G: promoter Tet-responsive element 3rd generation. rtTA 3G: DOX-inducible transactivator 3rd generation. PuroR: puromycin resistance gene. BlastiR: blasticidin resistance gene. hU6: human U6 promoter. mU6: mouse U6 promoter. ZF: zinc fingers.

Article Snippet: Donor plasmids used for knock-ins at the Rosa26 (pEN111) and Tigre locus (pEN366, Addgene #156432), as well as the Rosa26 - and Tigre -specific sgRNA-encoding plasmids (pX335-EN479, pX335-EN481 and pX330-EN1201 (Addgene #92144)) were all provided by E. Nora (UCSF).

Techniques: Transfection, Knock-In, Selection, Clone Assay, Expressing, Control, Zinc-Fingers