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 more than 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 more than 100,000 dual-gene perturbations. The polarized nature of spacer adaptation revealed the historical contingency in the stepwise acquisition of genetic perturbations leading to increasing antibiotic resistance. CALM circumvents the expense, labor, and time required for synthesis and cloning of gRNAs, allowing generation of CRISPRi libraries in wild-type bacteria refractory to routine genetic manipulation.