We present CRISPRware, an efficient method for generating guide RNA (gRNA) libraries against transcribed, translated, and noncoding regions. CRISPRware leverages next-generation sequencing data to design context-specific gRNAs and accounts for genetic variation, which allows allele-specific guide design on a genome-wide scale. The latter ability holds promise for the development of gene therapy in the context of gene dosing and dominant negative mutations.