CRISPRi pooled screening based functional genomics method in bacteria

As the rapid development of next generation sequencing (NGS), the ability to rapidly obtain the microbial genome at low cost is evolving rapidly. In contrast, due to the complexity of the genetic network, our understanding about the translation from the genome to phenotypes is still lacking and the conventional approach to study genes one by one cannot catch up with the step of boosting microbial genomes. Hence, it is badly needed to develop general functional genomics platform methods for bacteria enabling investigating genome-wide gene-phenotype association in a rapid and high-throughput manner.In this work, combining CRISPR interference technology that repressing gene expression at the transcriptional level, high-throughput DNA synthesis and sequencing methods, we successfully establish a novel functional genomics platform method for bacteria, fulfilling the requirements mentioned above. Briefly, we use microarray DNA synthesis method to prepare sgRNA library, which, plus CRISPRi tool, targets and represses every genes coded by a bacterial genome. Using NGS as a profiling method, the screening in selective conditions at pooled format enables investigating the impact of every gene encoded by a bacterial genome on the studied phenotype in very short time scale.Our results establish CRISPRi pooled screening as a powerful tool for mapping complex prokaryotic genetic networks to phenotypes in a precise, rapid and high-throughput manner. It enables dissecting the genome-scale profile towards multiple phenotypes in parallel, and is potent to better decode the translation from microbial genome to phenotypes.