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.
Key wordsCRISPR Cas9 gRNA Genome editing Transcription Repression
We are grateful to Donald L. Court (NCI-Frederick) for the generous gift of pSIM7 and to Dominick Matteau and Alain Lavigueur for critical reading of the manuscript. We thank the Centre de calcul scientifique of Université de Sherbrooke for computational resources and technical support. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). S.R. holds a Chercheur-boursier Junior 1 award from the Fonds de recherche Québec-Santé.