PAM-repeat associations and spacer selection preferences in single and co-occurring CRISPR-Cas systems

Abstract

Background The adaptive CRISPR-Cas immune system stores sequences from past invaders as spacers in CRISPR arrays and thereby provides direct evidence that links invaders to hosts. Mapping CRISPR spacers has revealed many aspects of CRISPR-Cas biology, including target requirements such as the protospacer adjacent motif (PAM). However, studies have so far been limited by a low number of mapped spacers in the database. Results By using vast metagenomic sequence databases, we map approximately one-third of more than 200,000 unique CRISPR spacers from a variety of microbes and derive a catalog of more than two hundred unique PAM sequences associated with specific CRISPR-Cas subtypes. These PAMs are further used to correctly assign the orientation of CRISPR arrays, revealing conserved patterns between the last nucleotides of the CRISPR repeat and PAM. We could also deduce CRISPR-Cas subtype-specific preferences for targeting either template or coding strand of open reading frames. While some DNA-targeting systems (type I-E and type II systems) prefer the template strand and avoid mRNA, other DNA- and RNA-targeting systems (types I-A and I-B and type III systems) prefer the coding strand and mRNA. In addition, we find large-scale evidence that both CRISPR-Cas adaptation machinery and CRISPR arrays are shared between different CRISPR-Cas systems. This could lead to simultaneous DNA and RNA targeting of invaders, which may be effective at combating mobile genetic invaders. Conclusions This study has broad implications for our understanding of how CRISPR-Cas systems work in a wide range of organisms for which only the genome sequence is known.

Transcriptional analysis of CRISPR I-B arrays of Leptospira interrogans serovar Lai and its processing by Cas6

“…Such inconsistency in the prediction of consensus PAM could be due to feeding different spacer lengths or its orientation during in silico analysis. Recently, a computational pipeline (Vink et al, 2021) has predicted the functional orientation of spacer and its associated PAM (5 -ATG-3 ) for the subtype I-B system of Leptospira, which agrees well with our results presented in this study and elsewhere (Prakash and Kumar, 2021).…”

A scaling law in CRISPR repertoire sizes arises from the avoidance of autoimmunity

“…First, we can extend our bioinformatic analysis of high-mismatch STSs (Figure S3) to a more detailed search for non-canonical PAMs, mutations in the adjoining repeat or cas locus, and the spacer being closest to the leader end of the array, which would provide additional evidence that high-mismatch self-targets cause autoimmunity and must be avoided. 12,39,40,67 Second, experiments can demonstrate that CRISPR-Cas subtypes with shorter spacers (e.g., types II-A and II-C) acquire spacers at a lower rate than those with longer spacers (e.g., types I-A, I-B, and III).…”

Adaptation by Type V-A and V-B CRISPR-Cas Systems Demonstrates Conserved Protospacer Selection Mechanisms Between Diverse CRISPR-Cas Types

“… 23 Interestingly, other type V subtypes do not contain either Cas1 or Cas2 and although it remains unclear how these systems acquire new spacers against new incoming MGEs, it seems most likely that co-occurring CRISPR-Cas systems provide this function. 24 , 35 , 47 …”