Structural analyses and engineering of Type IV-A CRISPR-Cas systems
Abstract
The constant evolutionary arms race between prokaryotes and phages has led to the development of a remarkable diversity of prokaryotic defense systems. Clustered regularly
interspaced short palindromic repeats (CRISPR) and their associated cas genes constitute an adaptive immune system that defends against mobile genetic elements (MGEs) such as phages
and plasmids. CRISPR-Cas mediated immunity typically relies on sequence specific target recognition and degradation of the invading nucleic acid. CRISPR-Cas systems are classified
into two classes either utilizing a multidomain effector protein in case of class 2 or an effector assembled of several proteins for target interference in class 1 systems. Type IV systems are
class 1 CRISPR-Cas systems usually lacking a nuclease for target cleavage. The subtype IV-A1 comprises the proteins Cas8, Cas7, Cas5, Cas6 and the CRISPR associated helicase DinG
(CasDinG), which together form an effector complex with an crRNA for gene regulation and plasmid clearance. Structural analyses of distinct type IV effectors were lacking, and type IV
CRISPR-Cas systems have not been adapted for genome engineering applications. Here, we first analyzed two type IV-A CRISPR-Cas effector complex structures using cryo
electron microscopy (cryo-EM) revealing how these complexes specifically recognize the protospacer adjacent motif (PAM) and target-strand DNA to form an R-loop structure. Further
analyses showed the interaction of the complex with the helicase CasDinG, whose translocation is essential for long-range transcriptional repression. With this study we provide the structural basis for engineering type IV-A-based genome editing tools. We expanded the available toolbox of CRISPR-Cas system functional in generating large
genomic deletions by a type I-Fv system and a type IV-A1 system. In the latter system, CasDinG was engineered into a helicase-nuclease fusion protein conferring DNA cleavage activity. Using whole genome sequencing (WGS), we analyzed the genomic deletions in Escherichia coli showing long-range deletions of almost 44 kb and 46 kb for the engineered type IV-A1 and
type I-Fv, respectively. These deletions terminated at an inverted repeat sequence of an IS1 element indicating its associated in DNA repair. Further, small deletions of 51-255 bp were
observed flanked by several homologous base pairs suggesting microhomology-mediated repair of the CRISPR-induced DNA lesions. Together, this research contributes to a better understanding of CRISPR-Cas type IV-A1 systems, highlighting their unique interference mechanisms and their potential as tool for biotechnological approaches.
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