Developing the CRISPR Interference System to Understand Bacterial Gene Function
Gene function in bacteria is typically studied by isolation and characterization of mutants with loss of gene function. Since isolation or construction of specific mutants can be problematic when studying multiple genes with redundant function or bacteria with poorly developed genetic systems, we have developed an alternative genetic approach to study gene function. For this, we have exploited the observation that CRISPR (clustered regularly interspaced short palindromic repeats), which normally serves to protect bacterial cells against transfer of foreign DNA, has been modified as a gene silencing system. We have improved the use of a mutationally altered Cas9 protein [Qi, L], along with co-expression of an RNA guide molecule, to repress expression of a number of genes in E. coli K-12 strains. This system has been used to silence expression of selected genes. This research should lead to a better understanding of gene function, as well as a general method to study pathogenic bacteria in their natural hosts.