CRISPR/Cas9 is a powerful genetic engineering technology that enables the introduction of genomic changes such as deletions and insertions of specific bits of DNA in cells with high precision. Compared to other programmable DNA nuclease such as ZFNs and TALENs, the specific binding of the Cas9 nuclease is mediated by a small guide RNA (gRNA), which can easily be designed to target any locus in the genome. The ease of generating novel gRNA vectors and its high efficiency has rapidly made CRISPR-Cas9 the dominant tool in gene editing applications, including gene knockout, knockin, tagging, etc. Here we describe our method for rapid and efficient generation of gene knockout or deletion cells using CRISPR/Cas9 within the time span of one month. The design of gRNAs, plasmid cloning, transfection, cell culturing, positive clone selection, and screening can be obtained from this method.
Keywords: DNA; Deletion; Endonuclease; Gene editing; Genomic engineering; Transfection; gRNA.