Dec 18, 2020 · In the present study, we developed SUCCESS to generate KO clones using two plasmids coding Cas9 and gRNA, two 80mer ssODNs, and a blunt-end universal cassette with selection marker.

In this review, we detail the molecular mechanisms of the CRISPR/Cas9 system when using Cas9 from Streptococcus pyogenes (Sp Cas9; hereinafter referred to as Cas9) for knock-in approaches and explore the current strategies attempted to increase their efficiency in mammalian cells.

Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment.

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 · CRISPR/Cas9 technology enables the rapid generation of loss-of-function mutations in a targeted gene in mammalian cells. A single cell harboring those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone.

Jul 1, 2024 · Several strategies are available for enhancing the efficiency and precision of CRISPR/Cas9 knockout, and these involve the consideration of (1) the number of sgRNAs transfected into cell lines and (2) the type of CRISPR/Cas9: plasmid, mRNA, and protein.

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. Our method, optimized for a wide spectrum of cancer cell lines, makes CRISPR more accessible for applications in personalized and precision medicine.

Dec 9, 2021 · Here we designed and characterized a knockout fragment intended to repair Cas9-induced gene disruptions by homologous recombination. We identified knockout clones of Komagataella phaffii with high fidelity by PCR, removing the need for Sanger sequencing.

1 day ago · We employed a CRISPR/Cas9 technique in Leishmania major to evaluate its efficiency in editing a kDNA-associated gene, the universal minicircle sequence binding protein (UMSBP), which is involved in mitochondrial respiration and kinetoplast division. Using this toolkit, we generated UMSBP mNG-tagged and single knockout L. major (LmUMSBP+/−) parasites, which were confirmed by PCR, confocal ...

4 days ago · Hepatocellular carcinoma (HCC) resists immunotherapy due to its immunosuppressive microenvironment. Sarcoma homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) inhibits T cell receptor signalling, and its pharmacological inhibition is limited by poor selectivity and membrane permeability. Here, we generated CRISPR-edited SHP-1-knockout (KO) CD8+ T cells to enhance adoptive ...