Bacteria from the same species can differ widely in their gene content. In E. coli, the set of genes shared by all strains, known as the core genome, represents about half the number of genes present in any strain. While recent advances in bacterial genomics have unraveled genes required for fitness in various experimental conditions at the genome scale, most studies have focused on single model strains. As a result, the impact of this genetic diversity on core processes of the bacterial cell remains largely under-investigated. Here, we developed a new CRISPR interference platform for highthroughput gene repression that is compatible with most E. coli isolates and closely-related species. We applied it to assess the importance of ~3,400 nearly ubiquitous genes in 3 growth conditions in 18 representative E. coli strains spanning most common phylogroups and lifestyles of the species. Our screens revealed extensive variations in gene essentiality between strains and conditions. Investigation of the genetic determinants for these variations highlighted the importance of epistatic interactions with mobile genetic elements. In particular, we showed how prophage-encoded defense systems against phage infection can trigger the essentiality of persistent genes that are usually nonessential. This study provides new insights into the evolvability of gene essentiality and argues for the importance of studying various isolates from the same species under diverse conditions.