Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Pathogenic DBD mutants introduced into the endogenous BRCA2 gene cause cytosolic mis-localization and disable DNA repair by HDR. ( A ) Subcellular fractionation of BRCA2 mutant cell lines, HeLa-W2626C and HeLa-D2723H. Two independent clones for each mutation were used. Nuclear and cytoplasmic fractions of BRCA2 and RAD51 were analysed by western blotting. MEK2 is a cytoplasmic marker. ( B ) mClover Lamin A HDR assay. Cells transfected with Lamin A-targeting sgRNA and mClover Lamin A donor constructs as shown in the schematic were analysed for mClover Lamin A-positive cells. Mean of HDR positive cells (%) ± s.e. from two repeats is shown. More than 100 cells per sample were analysed in each repeat. Statistical significance was tested by one-way ANOVA test, followed by Bonferroni's multiple comparison test.

Article Snippet: Cells grown on coverslips in six-well plates were transfected with sgRNA plasmid targeting Lamin A (pUC CBA-SpCas9.EF1a-BFP.sgLMNA, Addgene Plasmid #98971) and donor plasmid (pCAGGS Donor mClover-LMNA, Addgene Plasmid #98970).

Techniques: Fractionation, Mutagenesis, Clone Assay, Western Blot, Marker, Transfection, Construct, Comparison

Journal: Nature communications

Article Title: Insights into the compact CRISPR-Cas9d system.

doi: 10.1038/s41467-025-57455-9

Figure Lengend Snippet: Fig. 3 | The hybrid REM of Cas9d comprising the REC domains and Stem 2 and stem 3 of the sgRNA. a Structural alignment of the Cas9d (RNA-coordinated target Engagement Module, REM) with the SpyCas9 REC domain. b Close-up view of the Cas9d REM, as highlighted by the ellipse in a. c Interaction interface of REC domain

Article Snippet: The gene encoding the full-length Cas9d (Sangon) was codon optimized for E.coli expression and assembled into a modified pET vector (2Bc-T, Addgene #37236) with a C-terminal thrombin-TwinStrepII-Histag usingGibson assembly (NewEnglandBiolabs, E2611L).Mutations in Cas9d and SpyCas9 (WT expression plasmid, Addgene #101199) were introduced using the QuickChange Mutagenesis kit (Takara, Cat# 638949) following the manufacturer’s instructions.

Techniques: Drug discovery

Journal: EMBO Reports

Article Title: Targeting monocytic Occludin impairs transendothelial migration and HIV neuroinvasion

doi: 10.1038/s44319-024-00190-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The initial plasmid backbone used was the pLenti-spCas9-T2A-Crimson-P2A-puro (kindly provided by Dr. Feng Zheng) and derived versions of this plasmid were also generated to express mNeonGreen (Addgene #122183), TagRFP-T (Addgene #122200) or iRFP670 (Addgene # 122182) fluorescent proteins.

Techniques: Isolation, Recombinant, Sequencing, Knock-Out, DNA Ligation, Transfection, Cell Viability Assay, Blocking Assay, Protease Inhibitor, Virus, Software, Electroporation, Imaging, Polymer, Microscopy, Flow Cytometry, Membrane

Journal: bioRxiv

Article Title: Engineering Nme2Cas9 Adenine Base Editors with Improved Activity and Targeting Scope

doi: 10.1101/2023.04.14.536905

Figure Lengend Snippet: a) Nme1Cas9/sgRNA/DNA ternary complex structure, PDB:6JDV. Nme2Cas9 is 98% identical to Nme2Cas9 outside of the WED and PAM-interacting domains. Black spheres represent N- and C-termini and colored spheres represent sites of domain insertion. Deaminase domain insertion sites (Nme2Cas9 aa numbers) are specified to the right, with colors matching the sites indicated in the structure. b) Activities of Nme2-ABE8e constructs in mCherry reporter cells (activated upon A-to-G editing) after plasmid transfection, measured by flow cytometry (n = 3 biological replicates in technical duplicate; data represent mean ± SD). c) A-to-G editing following transfection of Spy-ABE8e vs. Nme2-ABE8e plasmids, using PAM-matched, endogenous HEK293T genomic loci. The editing efficiency at the maximally edited adenine for each target was plotted. Editing efficiencies were measured by amplicon deep sequencing (n = 3 biological replicates; data represent mean ± SD). d) Data from (c) were aggregated and replotted, with each data point representing the maximum A-to-G editing efficiency of an individual target site, as measured by amplicon deep sequencing (n = 3 biological replicates; data represent mean ± SEM). e) Summary of mean A-to-G editing activities and editing windows for Spy- and Nme2-ABE8e constructs in HEK293T cells. Numbers provided for each position in the protospacer represent the mean A-to-G editing efficiency across eight PAM-matched endogenous target sites, as measured via amplicon deep sequencing (n = 3 biological replicates). Crossed-out boxes indicate that no adenine was present at the specified position in the target panel tested.

Article Snippet: U6-driven sgRNA plasmids for the various Cas effectors were cloned using pBluescript sgRNA expression plasmids (Addgene #122089, #122090, #122091 for SpyCas9, SauCas9 and Nme2Cas9 respectively).

Techniques: Construct, Plasmid Preparation, Transfection, Flow Cytometry, Amplification, Sequencing

Journal: Oncotarget

Article Title: Mutation of the Sp1 binding site in the 5′ flanking region of SRY causes sex reversal in rabbits

doi: 10.18632/oncotarget.16979

Figure Lengend Snippet: Generation of genetically targeted SRY-sp1-KO rabbits using CRISPR/Cas9 system

Article Snippet: Two complementary DNA oligonucleotides were annealed at 95°C for 5 min to generate double-strand DNA then cloned into a BbsI-digested pUC57-simple vector expressing Cas9 (Addgene ID 51307 for cells and Addgene ID 51307 for zygotes).

Techniques: CRISPR, Injection