Diversity of CRISPR-Cas immune systems and molecular machines

Abstract

Bacterial adaptive immunity hinges on CRISPR-Cas systems that provide DNA-encoded, RNA-mediated targeting of exogenous nucleic acids. A plethora of CRISPR molecular machines occur broadly in prokaryotic genomes, with a diversity of Cas nucleases that can be repurposed for various applications.

Fig. 1

CRISPR-Cas systems and adaptive immunity. CRISPR repeats, together with CRISPR spacers, constitute repeat-spacer arrays that define clustered regularly interspaced short palindromic repeats (CRISPRs). These CRISPR arrays are typically flanked by CRISPR-associated sequences (cas) that encode Cas proteins involved in the three stages of CRISPR-encoded immunity, namely adaptation, expression and interference. During adaptation, Cas proteins (including the universal Cas1 and Cas2) sample invasive DNA, leading to the genesis of a new repeat-spacer unit that is inserted in a polarized manner in the CRISPR array. During the second stage — expression — the CRISPR array is transcribed into a full pre-crRNA transcript that is processed into small, mature, interfering CRISPR RNAs (crRNAs). In the third — interference — stage, crRNAs guide Cas effector proteins towards complementary nucleic acids for sequence-specific targeting. Interaction between the interference complex and the target nucleic acid is typically initiated by binding to the protospacer adjacent motif (PAM), which triggers interrogation of flanking DNA by the loaded crRNA. If complementarity extends beyond the seed sequence, an R-loop is formed, and nickase domains within Cas effector proteins cleave the target DNA. dsDNA double-stranded DNA, L leader

Fig. 2

Diversity of CRISPR-Cas molecular machines. Two main classes of CRISPR-Cas systems exist, which are defined by the nature of their Cas effector nucleases, either constituted by multiprotein complexes (class 1), or by a single signature protein (class 2). For class 1 systems, the main types of CRISPR-Cas systems include type I and type III systems. Illustrated here as an example, the Escherichia coli K12 type I-E system (upper left) targets sequences flanked by a 5′-located PAM. Guide RNAs are generated by Cascade, in a Cas6-defined manner and typically contain an eight-nucleotide 5′ handle derived from the CRISPR repeat, a full spacer sequence, and a 3′ hairpin derived from the CRISPR repeat. Following nicking of the target strand, the 3′ to 5′ Cas3 exonuclease destroys the target DNA in a directional manner. In the Pyrococcus furiosus DSM 3638 type III-B system (lower left), a short crRNA guide directs the Cmr complex towards complementary single-stranded RNA in a PAM-independent manner. For the canonical type II-A Streptococcus thermophilus LMD-9 system (upper right), a dual crRNA–tracrRNA guide generated by Cas9 and RNase III targets a 3′-flanked PAM DNA complementary sequence for the genesis of a precise double-stranded break using two nickase domains (RuvC and HNH). For the Francisella novicida U112 type V system (lower right), a single guide RNA targets complementary dsDNA flanked by a 5′-PAM using Cpf1, which generates a staggered dsDNA break. Cascade CRISPR-associated complex for antiviral defense, CRISPR clustered regularly interspaced short palindromic repeat, crRNA CRISPR RNA, dsDNA double-stranded DNA, L leader, nt nucleotide, PAM protospacer adjacent motif, ssRNA single-stranded RNA, tracrRNA trans-activating CRISPR RNA

Fig. 3

Applications and targets of CRISPR-Cas systems. CRISPR-Cas systems can target various types of nucleic acids, including invasive and mobile DNA (green box), or endogenous sequences (blue box). In their native environment, CRISPR-Cas systems naturally target mobile and exogenous DNA elements. Conversely, engineered systems are typically designed to target self-DNA to trigger endogenous modifications. Targeting can be directed at bacteriophage DNA to provide anti-viral defense (upper left). Likewise, Cas nucleases can be directed at plasmid DNA in order to prevent uptake and dissemination of undesirable sequences or to cure the host of plasmid sequences (center left). Targeting can also be directed at mobile DNA elements such as transposons so as to maintain DNA integrity and ensure homeostasis (lower left). When aiming the CRISPR-Cas machinery towards the cell’s own chromosomal content, the purpose is typically to induce endogenous DNA repair pathways to drive editing of the DNA sequence (upper center). Catalytically deactivated variants of Cas nucleases can be used as DNA-binding proteins to block transcription (CRISPRi, upper right), or can be fused to transcriptional activators to activate transcription (CRISPRa, center right). Alternatively, Cas nucleases can be reprogrammed to trigger a lethal auto-immune response, leading to cell death (bottom right). CRISPR sequences themselves can be used for genotyping, by using the series of vaccination events as a genetic historical record (lower center). Cas CRISPR associated, CRISPR clustered regularly interspaced short palindromic repeat, CRISPRa CRISPR activation, CRISPRi CRISPR interference

Fig. 4

Exploitation of endogenous and engineered CRISPR-Cas systems in bacteria. Exogenous DNA sequences can be targeted by CRISPR-Cas systems to build up phage resistance in food starter cultures (to vaccinate yoghurt strains against bacteriophage), to prevent the uptake and dissemination of plasmids that encode undesirable traits such as antibiotic resistance genes (to immunize probiotic strains used in dietary supplements), or to ensure the genetic integrity and genomic homeostasis of valuable cultures (to fend off mobile genetic elements such as transposons and prophages) (upper panels). Unique records of iterative vaccination events captured as a series of spacers in CRISPR arrays can be used as sequencing targets for the detection, monitoring and typing of strains of interest, which include food cultures, spoilage organisms or pathogens (center panels). By contrast, self-targeting and engineered applications can be used in industrial settings to improve industrial workhorses by genome editing (indicated by ‘scissors’ symbol), or by re-directing the metabolic flux of various pathways for synthetic and yield purposes (lower panels). Lethal self-targeting can also be harnessed for the select eradication of pathogens or contaminants of interest. CRISPRa CRISPR activation, CRISPRi CRISPR interference