High-throughput profiling of sgRNA activities in CRISPR/Cas9 genome editing in bacteria

CRISPR/Cas9 is a promising tool in prokaryotic genome engineering, but its success is limited by the widely varying on-target activity of single guide RNAs (sgRNAs). Based on the association of CRISPR/Cas9-induced DNA cleavage with cellular lethality, we systematically profiled sgRNA activity by co-expressing a genome-scale library (~70,000 sgRNAs) with Cas9 or its specificity-improved mutant in Eecherichia coli. Based on this large-scale dataset, we constructed a comprehensive and high-density sgRNA activity map, which enables selecting highly active sgRNAs for any locus across the genome in this model organism.