engleski

Curiosities of Naïve and Primed CRISPR Adaptation in Escherichia Coli

To protect themselves from invasive mobile genetic elements (MGE), prokaryotes have developed many defense strategies. The CRISPR-Cas adaptive immune system allows prokaryotes to defend themselves by memorizing, recognizing and destroying MGEs. The memory is acquired through integration of a small piece of foreign DNA (spacer) into the CRISPR locus of invaded bacteria by the Cas1-Cas2 complex. This is known as ‘naïve CRISPR adaptation’. In next steps, CRISPR locus is transcribed and processed into CRISPR RNA (crRNA) which, guided by Cascade complex, base-pairs to target DNA. Upon recognition, DNA is degraded by the nuclease/helicase Cas3. In addition to DNA target recognition, Cascade can promote the acquisition of new spacers from invasive DNA, a response called ‘primed adaptation’ or ‘priming’. In E. coli priming requires all components of the CRISPR-Cas system - Cas3 protein, Cascade complex and Cas1-Cas2 proteins. In our previous work in E. coli we showed that naïve adaptation needs help from the host cell genome stability proteins to prepare suitable DNA substrates for integration. In several rec mutants we observed unusual pattern of naïve adaptation which could be explained by the possible lethality of the spacer integration process. We also found that Cas3 protein changes its conformation and activity at 37 °C suggesting that temperature may also have a role in priming. In this work we further investigated how growth and other host factors affect naïve and primed adaptation in E. coli. We will present current results and progress in understanding naïve and primed adaptation.

CRISPR-Cas ; Cas1, Cas2 ; adaptation, E. coli

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