CRISPR/Cas9-mediated Gene KnockoutGenomics
CRISPR technology has become a commonly used tool for site-directed genome editing in various biological systems. It is an easy, versatile and robust technique for conducting gene knockout and studying gene function. The isolation of successfully edited single-cell colonies depends not only on the quality of cell preparation and the efficiency of transfection but also on the accurate execution and annotation of critical liquid handling steps such as serial dilutions.
After the transfection of vectors coding for guide RNA and Cas9 protein, a sequence-specific double-strand break is generated (Figure 1). The broken sites are subsequently repaired by the endogenous cellular NHEJ machinery leading to an Indel mutation, which in many cases results in gene knockout. Candidate clones grown from single cells are isolated and screened to identify those containing the mutation. Finally, DNA sequencing of the mutant allele(s) is performed for positive clones.
Figure 1: Site-specific double-strand break of the target DNA sequence mediated by the CRISPR-Cas9 machinery/.
Original image from From technical note, Genome Editing: Which Should I Choose, TALEN or CRISPR by Ed Davis, Ph.D., Genecopoeia.
Watch the video tutorial about gene knockout experiment using CRISPR-Cas9 technology