cas9 expression vector pst1374 cas9  (Addgene inc)

Journal: bioRxiv

Article Title: A CRISPR/Cas9-based system using dual-sgRNAs for efficient gene deletion in Mycobacterium abscessus

doi: 10.1101/2025.05.28.656731

Figure Lengend Snippet: (A) Schematic of CRISPR-Cas9-induced gene deletions using dual sgRNAs. M. abscessus cells harboring pCas9-mScarlet and pQL033-Xsg plasmids express Sth1Cas9 and dual sgRNAs upon aTc induction. The sgRNAs guide Sth1Cas9 to the target genomic loci, inducing double-stranded DNA breaks that can result in precise deletions, single indels, double indels, or inaccurate deletions depending on the repair mechanism. (B) Dual-plasmid CRISPR/Cas9 workflow. M. abscessus is first transformed with the integrative plasmid pCas9-mScarlet (KanR) encoding inducible Cas9. This Cas9-expressing strain is then transformed with pQL033-Xsg, a plasmid carrying the dual-sgRNA cassette and ZeoR selection marker. CRISPR system activation results in gene deletion and loss of function.

Article Snippet: The Cas9 expression plasmid was derived from pLJR962 (Addgene #115162) via site-directed mutagenesis to introduce A9D and A599H mutations, restoring nuclease activity and generating pCas9.

Techniques: CRISPR, Plasmid Preparation, Transformation Assay, Expressing, Selection, Marker, Activation Assay

Journal: iScience

Article Title: Epigenomic analysis identifies DTP subpopulation using HOPX to develop targeted therapy resistance in lung adenocarcinoma

doi: 10.1016/j.isci.2025.112387

Figure Lengend Snippet: Deletion of HOPX impairs the progress of DTP regrowth (A) Western blot showing reduction of HOPX expression in PC9 cells by CRISPR-Cas9 mediated deletion of HOPX gene. (B) Cell growth curve of PC9 cells transfected with sgRNAs targeting HOPX or non-target sgRNAs. (C) Cell viability of PC9 cells transfected with sgRNAs targeting HOPX or non-target sgRNAs under the treatment of 150nM osimertinib at indicated time points (normalized to day0). Right: Bar graph showing cell viability at day 28 ( n = 4, including duplicates for sgRNA1 and duplicates for sgRNA2). (D) Colony formation assay on PC9 cells, at seeding density of 150,000 cells/well under 150nM osimertinib starting at 24 h after seeding. Left: Two representative images taken at 20 days after treatment per condition. Right: Quantification of colony numbers based on the representative fields shown on the left ( n = 4), including duplicates for sgRNA1 and duplicates for sgRNA2. (E) Western blot showing reduction of HOPX expression in NCI-H358 cells. (F) Cell growth of NCI-H358 cells transfected with sgRNAs targeting HOPX or non-target sgRNAs. (G) Cell viability of NCI-H358 cells transfected with sgRNAs targeting HOPX or non-target sgRNAs under the treatment of 500nM sotorasib at indicated time points (normalized to day 0). Right: Bar graph showing cell viability at day 25 ( n = 4, including duplicates for sgRNA1 and duplicates for sgRNA2). (H) Colony formation assay on NCI-H358 cells, at seeding density of 150,000 cells/well under 500nM sotorasib starting at 24 h after seeding. Left: Two representative images taken at 20 days after treatment per condition. Right: Quantification of colony numbers based on the representative fields shown on the left ( n = 4), including duplicates for sgRNA1 and duplicates for sgRNA2. Data are presented as means ± S.D. ( n = 4). Statistical significance was determined using Mann-Whitney test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Cells stably expressing human codon-optimized S. pyogenes Cas9 expression were generated by infection with the lentiCas9-Blast plasmid (a gift from Feng Zhang, Addgene #52962). sgRNAs targeting HOPX (selected from Brunello library) and non-target sgRNAs (selected from the Gecko library v2) were synthesized by Integrated DNA Technologies and listed in sgRNAs were cloned into lentiGuide-Puro (Addgene #52963) at Esp3I site.

Techniques: Western Blot, Expressing, CRISPR, Transfection, Colony Assay, MANN-WHITNEY