CRISPR/Cas9-mediated targeted mutagenesis in upland cotton (Gossypium hirsutum L.)
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is an adaptive immune system that exists in a variety of microbes. It could be engineered to function in eukaryotic cells as a fast, low-cost, efficient, and scalable tool for manipulating genomic sequences. In this chapter, detailed protocols are described for harnessing the CRISPR-Cas9 system from Streptococcus pyogenes to enable RNA-guided genome engineering applications in mammalian cells. We present all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs (gRNAs) and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination. These tools provide researchers with new instruments that accelerate both forward and reverse genetics efforts.