In bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system against phages and other foreign ...genetic elements. Here, we review the biology of the diverse CRISPR-Cas systems and the major progress achieved in recent years in understanding the underlying mechanisms of the three stages of CRISPR-Cas immunity: adaptation, crRNA biogenesis, and interference. The ecology and regulation of CRISPR-Cas in the context of phage infection, the roles of these systems beyond immunity, and the open questions that propel the field forward are also discussed.
CRISPR-Cas systems provide bacteria and archaea with diverse mechanisms of adaptive immunity, illuminating the evolutionary pressures imposed by selfish genetic elements.
Abstract
Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, ...the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms' efficacy on small and large external datasets, and provide a versatile combined on- and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs.
CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA. The well-studied types I, II and ...III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA) and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA. In type II systems, RNase III cleaves pre-crRNA base-paired with trans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA-crRNA duplex then guides Cas9 to cleave target DNA. Here, we demonstrate a novel mechanism in CRISPR-Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5'-YTN-3' protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5' overhang. The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR-Cas systems so far described.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR ...RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
The number and diversity of known CRISPR-Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR-Cas systems and cas genes, with an ...emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR-Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR-Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR-Cas.
Eric Lander, Françoise Baylis, Feng Zhang, Emmanuelle Charpentier, Paul Berg and specialists from seven countries call for an international governance framework.
Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA ...during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.
CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence ...foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans-encoded small RNA with 24-nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.
Prevotella spp. are a dominant bacterial genus within the human gut. Multiple Prevotella spp. co-exist in some individuals, particularly those consuming plant-based diets. Additionally, Prevotella ...spp. exhibit variability in the utilization of diverse complex carbohydrates. To investigate the relationship between Prevotella competition and diet, we isolated Prevotella species from the mouse gut, analyzed their genomes and transcriptomes in vivo, and performed competition experiments between species in mice. Diverse dominant Prevotella species compete for similar metabolic niches in vivo, which is linked to the upregulation of specific polysaccharide utilization loci (PULs). Complex plant-derived polysaccharides are required for Prevotella spp. expansion, with arabinoxylans having a prominent impact on species abundance. The most dominant Prevotella species encodes a specific tandem-repeat trsusC/D PUL that enables arabinoxylan utilization and is conserved in human Prevotella copri strains, particularly among those consuming a vegan diet. These findings suggest that efficient (arabino)xylan-utilization is a factor contributing to Prevotella dominance.
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•Metagenomics and culturomics characterization of Prevotella spp. in the mouse gut•Distinct Prevotella spp. compete in vivo for similar plant-derived polysaccharides•A dominant Prevotella strain encodes a specific PUL enabling arabinoxylan utilization•Similar genetic features are found in human Prevotella copri strains enriched in vegans
Prevotella are among the most dominant bacteria found in the gut. Galvez et al. identify Prevotella species in the mouse gut and examine how one dominant species outcompetes commensal bacteria, including other Prevotella species, by encoding specific polysaccharide utilization loci (PULs), notably those enabling arabinoxylan utilization.
RNA degradation is an essential process that allows bacteria to control gene expression and adapt to various environmental conditions. It is usually initiated by endoribonucleases (endoRNases), which ...produce intermediate fragments that are subsequently degraded by exoribonucleases (exoRNases). However, global studies of the coordinated action of these enzymes are lacking. Here, we compare the targetome of endoRNase Y with the targetomes of 3'-to-5' exoRNases from Streptococcus pyogenes, namely, PNPase, YhaM, and RNase R. We observe that RNase Y preferentially cleaves after guanosine, generating substrate RNAs for the 3'-to-5' exoRNases. We demonstrate that RNase Y processing is followed by trimming of the newly generated 3' ends by PNPase and YhaM. Conversely, the RNA 5' ends produced by RNase Y are rarely further trimmed. Our strategy enables the identification of processing events that are otherwise undetectable. Importantly, this approach allows investigation of the intricate interplay between endo- and exoRNases on a genome-wide scale.
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