Comprehensive genome-wide perturbations via CRISPR adaptation reveal complex genetics of antibiotic sensitivity

Genome-wide CRISPR screens enable systematic interrogation of gene function. However, guide-RNA libraries are costly to synthesize and their limited diversity compromises the sensitivity of CRISPR screens. Using the Streptococcus pyogenes CRISPR-Cas adaptation machinery, we developed CRISPR-adaptation-mediated library manufacturing (CALM), which turns bacterial cells into “factories” for generating hundreds of thousands of crRNAs covering 95% of all targetable genomic sites. With an average gene targeted by >100 distinct crRNAs, these highly comprehensive CRISPRi libraries produced varying degrees of transcriptional repression critical for uncovering novel antibiotic resistance determinants. Furthermore, by iterating CRISPR adaptation, we rapidly generated dual-crRNA libraries representing >100,000 dual-gene perturbations. The polarized nature of spacer adaptation revealed the historical contingency in the step-wise acquisition of genetic perturbations leading to increasing antibiotic resistance. CALM circumvents the expense, labor, and time required for synthesis and cloning of gRNAs, thus allowing generation of CRISPRi libraries in wild-type bacteria refractory to routine genetic manipulation.