About this video

• Video Title: Gene Knockout using CRISPR
• Channel: OriGene Technologies Inc.
• Speakers: None explicitly named.
• Duration: 00:07:36

Overview

This video explains the process of gene knockout using CRISPR technology. It details how CRISPR simplifies gene function studies by enabling targeted disruption of genes, making it more accessible and less resource-intensive than conventional methods. The video outlines the mechanisms of DNA repair after CRISPR-induced breaks and presents a specific protocol for CRISPR knockout and knock-in using OriGene's kits, including experimental steps, controls, and verification methods.

Key takeaways

• Gene Knockout for Function Studies: Gene knockout is a technique to understand gene function by observing the phenotype after disrupting a specific gene.
• CRISPR Technology Advancement: CRISPR has made gene knockout more accessible by enabling double-stranded breaks at specific genomic sites, overcoming the time, cost, and labor limitations of traditional animal models.
• DNA Repair Mechanisms: After CRISPR-induced double-strand breaks, cells repair the DNA through either non-homologous end joining (NHEJ), which is error-prone and can lead to gene knockout, or homology-directed repair (HDR), which can incorporate desired genetic changes using a repair template.
• OriGene's CRISPR Knockout/Knock-in Kits: These kits utilize the HDR system for gene knockout and knock-in. They provide an all-in-one vector for the gRNA and Cas9 protein, and a separate donor vector containing a reporter cassette (GFP and puromycin resistance) flanked by homologous arms, facilitating gene replacement and selection of edited cells.
• Experimental Protocol: The process involves co-transfection of gRNA and donor vectors, followed by cell passaging for approximately 20 days to allow for the loss of episomal DNA and integration of the donor vector. Puromycin selection is then applied to isolate successfully edited cells, which can be further analyzed using Western blotting or genomic PCR and sequencing.