Search

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.

Among the multiple antiviral defense mechanisms found in prokaryotes, CRISPR-Cas systems stand out as the only known RNA-programmed pathways for detecting and destroying bacteriophages and plasmids. Class 1 CRISPR-Cas systems, the most widespread and diverse of these adaptive immune systems, use an RNA-guided multiprotein complex to find foreign nucleic acids and trigger their destruction. In this review, we describe how these multisubunit complexes target and cleave DNA and RNA and how regulatory molecules control their activities. We also highlight similarities to and differences from Class 2 CRISPR-Cas systems, which use a single-protein effector, as well as other types of bacterial and eukaryotic immune systems. We summarize current applications of the Class 1 CRISPR-Cas systems for DNA/RNA modification, control of gene expression, and nucleic acid detection.

Genetic conflict between viruses and their hosts drives evolution and genetic innovation. Prokaryotes evolved CRISPR-mediated adaptive immune systems for protection from viral infection, and viruses have evolved diverse anti-CRISPR (Acr) proteins that subvert these immune systems. The adaptive immune system in Pseudomonas aeruginosa (type I-F) relies on a 350 kDa CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease for target degradation. Here, we report the cryo-electron microscopy (cryo-EM) structure of the Csy complex bound to two different Acr proteins, AcrF1 and AcrF2, at an average resolution of 3.4 Å. The structure explains the molecular mechanism for immune system suppression, and structure-guided mutations show that the Acr proteins bind to residues essential for crRNA-mediated detection of DNA. Collectively, these data provide a snapshot of an ongoing molecular arms race between viral suppressors and the immune system they target.

Archaea and bacteria harbour clustered regularly interspaced short palindromic repeats (CRISPR) loci. These arrays encode RNA molecules (crRNA), each containing a sequence of a single repeat–intervening spacer. The crRNAs guide CRISPR-associated (Cas) proteins to cleave nucleic acids complementary to the crRNA spacer, thus interfering with targeted foreign elements. Notably, pre-existing spacers may trigger the acquisition of new spacers from the target molecule by means of a primed adaptation mechanism. Here, we show that naturally occurring orphan CRISPR arrays that contain spacers matching sequences of the cognate (absent) cas genes are able to elicit both primed adaptation and direct interference against genetic elements carrying those genes. Our findings show the existence of an anti-cas mechanism that prevents the transfer of a fully equipped CRISPR–Cas system. Hence, they suggest that CRISPR immunity may be undesired by particular prokaryotes, potentially because they could limit possibilities for gaining favourable sequences by lateral transfer.

Archaea and bacteria harbour clustered regularly interspaced short palindromic repeats (CRISPR) loci. These arrays encode RNA molecules (crRNA), each containing a sequence of a single repeat–intervening spacer. The crRNAs guide CRISPR-associated (Cas) proteins to cleave nucleic acids complementary to the crRNA spacer, thus interfering with targeted foreign elements. Notably, pre-existing spacers may trigger the acquisition of new spacers from the target molecule by means of a primed adaptation mechanism. Here, we show that naturally occurring orphan CRISPR arrays that contain spacers matching sequences of the cognate (absent) cas genes are able to elicit both primed adaptation and direct interference against genetic elements carrying those genes. Our findings show the existence of an anti-cas mechanism that prevents the transfer of a fully equipped CRISPR–Cas system. Hence, they suggest that CRISPR immunity may be undesired by particular prokaryotes, potentially because they could limit possibilities for gaining favourable sequences by lateral transfer.

CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complementary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of challenges regarding Cas9 specificity, efficiency, fusion protein function, and spatiotemporal control within the cell remain. In this work, we develop a platform for constructing novel proteins to address these open questions. We demonstrate methods to either screen or select active Cas9 mutants and use the screening technique to isolate functional Cas9 variants with a heterologous PDZ domain inserted within the protein. As a proof of concept, these methods lay the groundwork for the future construction of diverse Cas9 proteins. Straightforward and accessible techniques for genetic editing are helping to elucidate biology in new and exciting ways; a platform to engineer new functionalities into Cas9 will help forge the next generation of genome-modifying tools.