Mar 16, 2023 · Knock-out (KO) cell lines help overcome the challenge of accessing the correct controls during assay development and enable confident interrogation of the relationship between genotype and phenotype.

Dec 2, 2019 · Gene knockout is a technique that selectively turns off a specific gene. Genes are important for us as they encode proteins. These proteins, later on, participate in different cellular activities and metabolic pathways. Studying a gene’s function allows us to understand its role in cellular pathways and how it causes abnormalities.

Jun 17, 2021 · With its advantages – such as complete loss of gene function, regulation of any segment of the genome, and convenient recovery experiments - knockout cell lines are an irreplaceable tool in the research of disease mechanisms and drug targets.

Sep 20, 2024 · We describe steps for designing guide RNAs, cloning them into CRISPR-Cas9 vectors, cell seeding, transfection into cultured cells, clonal selection, and screening assays. This protocol can delete gene regions over 100 kbp, including GC-rich domains, and is applicable to various cell lines.

Jul 26, 2019 · Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation of true knockout clones and are amenable to multiplexed gene targeting.

In this study we describe an optimized method to efficiently create stable cell lines for functional studies. This method was successfully used to generate a PIEZO1 gene-KO cell line with the CRISPR/Cas9 technology, and TRPC5/GCaMP6f-mCherry-coexpressing cell lines without antibiotic selection.

Aug 13, 2024 · High knockout efficiencies (80-90% KO frequency) can now be quickly and inexpensively generated in a heterogeneous population of cells, referred to as a Knockout Cell Pool, allowing researchers to either directly assay the edited cell pool or generate clonal cell lines as quickly as possible.

Nov 14, 2024 · Using this method, we successfully created several new generations of colorectal cancer cell lines with monoallelic and biallelic knockouts of the epithelial cell adhesion molecule (EpCAM) gene.

May 20, 2021 · We can perform a variety of knockout (KO) strategies to generate a custom cell line model, including frameshift mutation, large fragment knockout, and multiple genes knockout.

We demonstrate the broad utility of this approach by transferring a functional, pathogenic mutation in the CTNNB1 gene from one colorectal cancer cell line to another.