Fidelity of prespacer capture and processing is governed by the PAM-mediated interactions of Cas1-2 adaptation complex in CRISPR-Cas type I-E system

Abstract

Edited by Joel M. Gottesfeld Prokaryotes deploy CRISPR-Cas-based RNA-guided adaptive immunity to fend off mobile genetic elements such as phages and plasmids. During CRISPR adaptation, which is the first stage of CRISPR immunity, the Cas1-2 integrase complex captures invader-derived prespacer DNA and specifically integrates it at the leader-repeat junction as spacers. For this integration, several variants of CRISPR-Cas systems use Cas4 as an indispensable nuclease for selectively processing the protospacer adjacent motif (PAM) containing prespacers to a defined length. Surprisingly, however, a few CRISPR-Cas systems, such as type I-E, are bereft of Cas4. Despite the absence of Cas4, how the prespacers show impeccable conservation for length and PAM selection in type I-E remains intriguing. Here, using in vivo and in vitro integration assays, deep sequencing, and exonuclease footprinting, we show that Cas1-2/I-E-via the type I-Especific extended C-terminal tail of Cas1-displays intrinsic affinity for PAM containing prespacers of variable length in Escherichia coli. Although Cas1-2/I-E does not prune the prespacers, its binding protects the prespacer boundaries from exonuclease action. This ensures the pruning of exposed ends by exonucleases to aptly sized substrates for integration into the CRISPR locus. In summary, our work reveals that in a few CRISPR-Cas variants, such as type I-E, the specificity of PAM selection resides with Cas1-2, whereas the prespacer processing is co-opted by cellular non-Cas exonucleases, thereby offsetting the need for Cas4.Prokaryotes utilize an adaptive immune response mediated by CRISPR and CRISPR-associated proteins (Cas) 2 to respond to infections by mobile genetic elements (MGE) (viz. phages and plasmids) (1-4). CRISPR encompasses a typical architec-This work was supported by Department of Biotechnology (DBT) Grants BT

Unraveling Exogenous DNA Processing in Cas4‐Lacking Crispr Systems: A Novel Bypass Pathway Explored

“…While certain type I-E CRISPR systems achieve adaptive immunity through the in vitro exonuclease activity displayed by Cas4 proteins, [14] many CRISPR-Cas systems lack Cas4 yet possess the ability to specifically and accurately select and process PAMs. [15] The mechanisms and structures guiding these systems in PAM Firstly, it involves ruler-guided modification and simultaneous PAM protection to modify exogenous DNA. Subsequently, the trimmed exogenous DNA becomes fully integrated into the host genome through two nucleophilic attacks.…”

“…While certain type I‐E CRISPR systems achieve adaptive immunity through the in vitro exonuclease activity displayed by Cas4 proteins, [ 14 ] many CRISPR‐Cas systems lack Cas4 yet possess the ability to specifically and accurately select and process PAMs. [ 15 ] The mechanisms and structures guiding these systems in PAM sequence selection and processing have remained elusive. A recent study has unveiled a breakthrough, indicating that the type I‐E CRISPR system accomplishes PAM selection and processing through the close collaboration between DEDDh and Cas1‐Cas2 complexes ( Figure 2 ).…”

A short overview of the CRISPR-Cas adaptation stage

“…As such, Cas4 prevents the integration of nonfunctional spacers with an inappropriate length or PAM (Kieper et al 2018;Lee et al 2018Lee et al , 2019Rollie et al 2018;Shiimori et al 2018;Almendros et al 2019). The function of Cas4 and DnaQ can also be performed by non-CRISPR nucleases in other systems (Yoganand et al 2019).…”

Digging into the lesser-known aspects of CRISPR biology

“…In the case of the I-E system of E. coli (devoid of Cas4 and with a Cas2 protein that does not exhibit exonuclease activity), DnaQ and ExoT exonucleases, as well as the proofreading subunit of the DNA polymerase III, have been suggested to be involved in the prespacer trimming (Kim et al 2020 ; Ramachandran et al 2020 ). According to the model proposed, once the prespacer is loaded into the Cas1-Cas2 complex and the PAM is recognised, host nucleases will degrade the ends of the prespacer until reaching the region protected by the complex (Yoganand et al 2019 ).…”