Abstract
Bacterial immune systems, such as CRISPR-Cas or restriction-modification (R-M) systems, affect bacterial pathogenicity and antibiotic resistance by modulating horizontal gene flow. A model system for CRISPR-Cas regulation, the Type I-E system from Escherichia coli, is silent under standard laboratory conditions and experimentally observing the dynamics of CRISPR-Cas activation is challenging. Two characteristic features of CRISPR-Cas regulation in E. coli are cooperative transcription repression of cas gene and CRISPR array promoters, and fast non-specific degradation of full length CRISPR transcripts (pre-crRNA). In this work, we use computational modeling to understand how these features affect the system expression dynamics. Signaling which leads to CRISPR-Cas activation is currently unknown, so to bypass this step, we here propose a conceptual setup for cas expression activation, where cas genes are put under transcription control typical for a restriction-modification (R-M) system and then introduced into a cell. Known transcription regulation of an R-M system is used as a proxy for currently unknown CRISPR-Cas transcription control, as both systems are characterized by high cooperativity, which is likely related to similar dynamical constraints of their function. We find that the two characteristic CRISPR-Cas control features are responsible for its temporally-specific dynamical response, so that the system makes a steep (switch-like) transition from OFF to ON state with a time-delay controlled by pre-crRNA degradation rate. We furthermore find that cooperative transcription regulation qualitatively leads to a cross-over to a regime where, at higher pre-crRNA processing rates, crRNA generation approaches the limit of an infinitely abrupt system induction. We propose that these dynamical properties are associated with rapid expression of CRISPR-Cas components and efficient protection of bacterial cells against foreign DNA. In terms of synthetic applications, the setup proposed here should allow highly efficient expression of small RNAs in a narrow time interval, with a specified time-delay with respect to the signal onset.
Highlights
CRISPR-Cas are adaptive immune systems, which defend prokaryotic cells against foreign DNA, including viruses and plasmids
We here propose a model system for CRISPR-Cas induction by assuming that activation of crRNA production is put under transcriptional control exhibited in a restrictionmodification (R-M) immune system (Pingoud et al, 2014). As argued below, such model system would have qualitative features of transcription regulation expected for a CRISPR-Cas, and will keep the same transcript processing mechanism as that described for native system. This model system allows bypassing the currently unknown signaling that leads to CRISPRCas activation, and can be readily analyzed in silico, since transcription regulation of a well-studied R-M system (AhdI, see Bogdanova et al, 2008)—for which we previously showed that it can be reliably modeled—is used as a proxy for transcription regulation of CRISPR-Cas system
Putting cas genes under transcription control found in AhdI mimics the main qualitative features of CRISPRCas transcription regulation, namely, gradual synthesis of Cas proteins, cooperativity in transcription regulation, and putative autoregulation. Another advantage of this setup is that we previously showed that biophysical modeling can be used to:(i) explain in vitro measurements of the wild type and mutant R-M system transcription control (Bogdanova et al, 2008), (ii) explain in vivo measurements of the system dynamics (Morozova et al, 2015), (iii) effectively perturb the main R-M system features and relate these perturbations with the system dynamics (Rodic et al, 2017)
Summary
CRISPR-Cas are adaptive immune systems, which defend prokaryotic cells against foreign DNA, including viruses and plasmids. A CRISPR-Cas system consists of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) array and associated cas genes (Makarova et al, 2006; Barrangou et al, 2007; Brouns et al, 2008; Hille and Charpentier, 2016). Cas proteins take part in CRISPR adaptation, which is a process in which new spacers from viral genomes are inserted in CRISPR array. The cas genes and the CRISPR array are transcribed from separate promoters, which are located inside of the intergenic regions here denoted by IGLB and L (the leader sequence), respectively (see Figure 1; Pougach et al, 2010; Pul et al, 2010)
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