Selection and Validation of Spacer Sequences for CRISPR-Cas9 Genome Editing and Transcription Regulation in Bacteria

RNA-guided Cas9 nucleases derived from clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems have recently been adapted as sequence-programmable tools for various purposes such as genome editing and transcriptional regulation. A critical aspect of the system is the selection and validation of spacer sequences that allow precise targeting of the guide RNA-Cas9 complex. We describe a procedure involving computational and experimental steps to identify and test potentially interesting spacer sequences in bacterial genomes.