CRISPR/Cas9‐Mediated Gene Knockout in Cells and Tissues Using Lentivirus

Abstract CRISPR‐Cas9 has become a powerful and popular gene editing tool. However, successful application of this tool in the lab can still be quite daunting to many newcomers to molecular biology, mostly because it is a relatively lengthy process involving multiple steps with variations of each step. Here, we provide a reliable, stepwise, and newcomer‐friendly protocol to knock out a target gene in wild‐type human fibroblasts. This protocol involves sgRNA design using CRISPOR , construction of an “all‐in‐one” vector expressing both sgRNA and Cas9 using Golden Gate cloning, streamlined production of high‐titer lentiviruses in 1 week after molecular cloning, and transduction of cells to generate a knockout cell pool. We further introduce a protocol for lentiviral transduction of ex vivo mouse embryonic salivary epithelial explants. In summary, our protocol is useful for new researchers to apply CRISPR‐Cas9 to generate stable gene knockout cells and tissue explants using lentivirus. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1 : sgRNA design Basic Protocol 2 : Cloning sgRNA in plasmid vector containing Cas9 encoding sequence using golden gate cloning Basic Protocol 3 : Lentivirus packaging Basic Protocol 4 : Lentivirus transduction of cells Basic Protocol 5 : Lentivirus transduction of salivary gland epithelial buds