CRISPR-Cas immune systems are widespread in bacteria and archaea, but not ubiquitous. Previous work has demonstrated that CRISPR immunity is associated with an infection-induced fitness cost, which may help explain the patchy distribution observed. However, the mechanistic basis of this cost has remained unclear. Using Pseudomonas aeruginosa PA14 and its phage DMS3vir as a model, we perform a 30-day evolution experiment under phage mediated selection. We demonstrate that although CRISPR is initially selected for, bacteria carrying mutations in the phage receptor rapidly invade the population following subsequent reinfections. We then test three potential mechanisms for the observed cost of CRISPR: (1) autoimmunity from the acquisition of self-targeting spacers, (2) immunopathology or energetic costs from increased cas gene expression and (3) toxicity caused by phage gene expression prior to CRISPR-mediated cleavage. We find that phages can express genes before the immune system clears the infection and that expression of these genes can have a negative effect on host fitness. While infection does not lead to increased expression of cas genes, it does cause differential expression of multiple other host processes that may further contribute to the cost of CRISPR immunity. In contrast, we found little support for infection-induced autoimmunological and immunopathological effects. Phage gene expression prior to cleavage of the genome by the CRISPR-Cas immune system is therefore the most parsimonious explanation for the observed phage-induced fitness cost.

CRISPR-Cas 免疫系统广泛存在于细菌和古生菌中，但并非无处不在。先前的研究表明，CRISPR 免疫与感染引起的适应度成本有关，这可能有助于解释观察到的斑块分布。然而，这种成本的机制基础仍不清楚。使用铜绿假单胞菌PA14及其噬菌体DMS3vir作为模型，我们在噬菌体介导的选择下进行了30天的进化实验。我们证明，尽管最初选择了 CRISPR，但携带噬菌体受体突变的细菌在随后的再感染后迅速侵入种群。然后，我们测试了观察到的 CRISPR 成本的三种潜在机制：(1) 获得自靶向间隔物产生的自身免疫，(2) cas基因表达增加导致的免疫病理学或能量成本，以及 (3) 噬菌体基因表达引起的毒性CRISPR介导的切割。我们发现噬菌体可以在免疫系统清除感染之前表达基因，并且这些基因的表达会对宿主的适应性产生负面影响。虽然感染不会导致表达增加cas基因，它确实会导致多个其他宿主过程的差异表达，这可能会进一步增加 CRISPR 免疫的成本。相比之下，我们几乎没有发现对感染诱导的自身免疫和免疫病理学影响的支持。因此，在 CRISPR-Cas 免疫系统切割基因组之前的噬菌体基因表达是对观察到的噬菌体诱导的适应性成本的最简单的解释。