Endogenous Gene Regulation as a Predicted Main Function of Type I-E CRISPR/Cas System in E. coli

Abstract

CRISPR/Cas is an adaptive bacterial immune system, whose CRISPR array can actively change in response to viral infections. However, Type I-E CRISPR/Cas in E. coli (an established model system), appears not to exhibit such active adaptation, which suggests that it might have functions other than immune response. Through computational analysis, we address the involvement of the system in non-canonical functions. To assess targets of CRISPR spacers, we align them against both E. coli genome and an exhaustive (~230) set of E. coli viruses. We systematically investigate the obtained alignments, such as hit distribution with respect to genome annotation, propensity to target mRNA, the target functional enrichment, conservation of CRISPR spacers and putative targets in related bacterial genomes. We find that CRISPR spacers have a statistically highly significant tendency to target i) host compared to phage genomes, ii) one of the two DNA strands, iii) genomic dsDNA rather than mRNA, iv) transcriptionally active regions, and v) sequences (cis-regulatory elements) with slower turn-over rate compared to CRISPR spacers (trans-factors). The results suggest that the Type I-E CRISPR/Cas system has a major role in transcription regulation of endogenous genes, with a potential to rapidly rewire these regulatory interactions, with targets being selected through naïve adaptation.

Cas3 Protein—A Review of a Multi-Tasking Machine

“…Regulatory roles for CRISPR systems are identified in several contexts [84][85][86]. The DNA targeting capabilities of Cascade, and observations that CRISPR systems frequently capture 'self' DNA from host chromosomes as well as 'non-self' invader DNA, indicate that CRISPR systems may regulate gene function as transcription factors [87,88]. Cascade gene targeting has been used for ectopic control of gene expression [89,90].…”

Systematic analysis of Type I‐E Escherichia coli CRISPR‐Cas PAM sequences ability to promote interference and primed adaptation

“…First, it does not exclusively target foreign DNA leading to acquisition of self-targeting spacers from cell’s own genome. Second, at least half of the spacers are selected from protospacers with dysfunctional PAMs (Yosef et al, 2012; Díez-Villaseñor et al, 2013; Levy et al, 2015; Bozic et al, 2019) and resulting crRNAs are not interference-proficient.…”

A Simple Criterion for Inferring CRISPR Array Direction

“…To address this computationally, one has to systematically examine CRISPR spacer (i.e., the corresponding crRNAs) interactions with host genome sequences. Either dsDNA [as experimentally found in II-B system of F. novicida (Ratner et al, 2019), and also computationally predicted for I-E in E. coli (Bozic et al, 2019)], or mRNA [as in I-F and I-C systems from, respectively, Pseudomonas aeruginosa and Campylobacter jejuni (Li et al, 2016; Dugar et al, 2018)] can be targeted in CRISPR/Cas non-canonical functions. Moreover, in canonical functions, the system can also target either dsDNA (in Type I and II) or mRNA (in Type III), as schematically shown in Figure 1A.…”

“…Experimental evidence that CRISPR/Cas systems that belong to other subtypes (e.g., Type II-C, Type I-F), are also exhibiting non-canonical functions through different functional/mechanistic modalities, are now accumulating (Veesenmeyer et al, 2014; Li et al, 2016; Dugar et al, 2018). From the computational side, we recently provided evidence (Bozic et al, 2019) that Type I-E CRISPR/Cas system from Escherichia coli has a clear preference to target host bacterial sequences vs. more than 230 sequenced E. coli phages. The predicted distribution of crRNA targets in the host genome is highly non-random, with the preference to target transcriptionally active regions and dsDNA rather than mRNA sequences.…”

“…Therefore, knowing the array orientation allows assessing crRNA interactions with sense vs. antisense DNA strand, and consequently separating bona fide targets from false positives. Likewise, when prior knowledge of the nature of the CRISPR-target is missing, as for the E. coli I-E system that we recently analyzed (Bozic et al, 2019), the array orientation enables assigning the underlying regulatory modality (dsDNA vs. mRNA targeting). The information on the CRISPR array orientation is indispensable even when crRNA is not the mediator of non-canonical activities, as in II-B system of F. novicida , where a duplex of small accessory RNAs (scaRNA:tracrRNA, small CRISPR/Cas-associated RNA and trans-activating crRNA, respectively) binds the target.…”