ABSTRACT
Adaptive immunity of prokaryotes is mediated by CRISPR-Cas systems that employ a large variety of Cas protein effectors to identify and destroy foreign genetic material. The different targeting mechanisms of Cas proteins rely on the proper protection of the host genome sequence while allowing for efficient detection of target sequences, termed protospacers. A short DNA sequence, the protospacer-adjacent motif (PAM), is frequently used to mark proper target sites. Cas proteins have evolved a multitude of PAM-interacting domains, which enables them to cope with viral anti-CRISPR measures that alter the sequence or accessibility of PAM elements. In this review, we summarize known PAM recognition strategies for all CRISPR-Cas types. Available structures of target bound Cas protein effector complexes highlight the diversity of mechanisms and domain architectures that are employed to guarantee target specificity.
Subject(s)
CRISPR-Cas Systems/genetics , Nucleotide Motifs/genetics , Adaptation, Physiological/genetics , Autoimmunity/genetics , Base Sequence , Models, Molecular , Ribonucleases/metabolismABSTRACT
CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids. Type I CRISPR-Cas systems employ highly diverse, multi-subunit surveillance Cascade complexes that facilitate duplex formation between crRNA and complementary target DNA for R-loop formation, retention, and DNA degradation by the subsequently recruited nuclease Cas3. Typically, the large subunit recognizes bona fide targets through the PAM (protospacer adjacent motif), and the small subunit guides the non-target DNA strand. Here, we present the Apo- and target-DNA-bound structures of the I-Fv (type I-F variant) Cascade lacking the small and large subunits. Large and small subunits are functionally replaced by the 5' terminal crRNA cap Cas5fv and the backbone protein Cas7fv, respectively. Cas5fv facilitates PAM recognition from the DNA major groove site, in contrast to all other described type I systems. Comparison of the type I-Fv Cascade with an anti-CRISPR protein-bound I-F Cascade reveals that the type I-Fv structure differs substantially at known anti-CRISPR protein target sites and might therefore be resistant to viral Cascade interception.
Subject(s)
Bacterial Proteins/metabolism , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , DNA/metabolism , Endonucleases/metabolism , Nucleic Acid Heteroduplexes/metabolism , RNA, Guide, Kinetoplastida/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , CRISPR-Associated Proteins/chemistry , CRISPR-Associated Proteins/genetics , Crystallography, X-Ray , DNA/chemistry , DNA/genetics , Endonucleases/chemistry , Endonucleases/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Models, Molecular , Nucleic Acid Conformation , Nucleic Acid Heteroduplexes/chemistry , Nucleic Acid Heteroduplexes/genetics , Protein Binding , Protein Conformation , RNA Caps/metabolism , RNA, Guide, Kinetoplastida/chemistry , RNA, Guide, Kinetoplastida/genetics , Shewanella putrefaciens/enzymology , Shewanella putrefaciens/genetics , Structure-Activity RelationshipABSTRACT
Shewanella putrefaciens CN-32 contains a single Type I-Fv CRISPR-Cas system which confers adaptive immunity against bacteriophage infection. Three Cas proteins (Cas6f, Cas7fv, Cas5fv) and mature CRISPR RNAs were shown to be required for the assembly of an interference complex termed Cascade. The Cas protein-CRISPR RNA interaction sites within this complex were identified via mass spectrometry. Additional Cas proteins, commonly described as large and small subunits, that are present in all other investigated Cascade structures, were not detected. We introduced this minimal Type I system in Escherichia coli and show that it provides heterologous protection against lambda phage. The absence of a large subunit suggests that the length of the crRNA might not be fixed and recombinant Cascade complexes with drastically shortened and elongated crRNAs were engineered. Size-exclusion chromatography and small-angle X-ray scattering analyses revealed that the number of Cas7fv backbone subunits is adjusted in these shortened and extended Cascade variants. Larger Cascade complexes can still confer immunity against lambda phage infection in E. coli Minimized Type I CRISPR-Cas systems expand our understanding of the evolution of Cascade assembly and diversity. Their adjustable crRNA length opens the possibility for customizing target DNA specificity.
Subject(s)
Bacterial Proteins/chemistry , CRISPR-Associated Proteins/chemistry , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Escherichia coli/genetics , Shewanella putrefaciens/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacteriophage lambda/physiology , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/immunology , Escherichia coli/immunology , Escherichia coli/metabolism , Escherichia coli/virology , Gene Expression , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/immunology , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Alignment , Shewanella putrefaciens/immunology , Shewanella putrefaciens/metabolism , Shewanella putrefaciens/virology , Transformation, BacterialABSTRACT
Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs.
