Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System.

CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.

TemStaPro: protein thermostability prediction using sequence representations from protein language models.

MOTIVATION: Reliable prediction of protein thermostability from its sequence is valuable for both academic and industrial research. This prediction problem can be tackled using machine learning and by taking advantage of the recent blossoming of deep learning methods for sequence analysis. These methods can facilitate training on more data and, possibly, enable the development of more versatile thermostability predictors for multiple ranges of temperatures. RESULTS: We applied the principle of transfer learning to predict protein thermostability using embeddings generated by protein language models (pLMs) from an input protein sequence. We used large pLMs that were pre-trained on hundreds of millions of known sequences. The embeddings from such models allowed us to efficiently train and validate a high-performing prediction method using over one million sequences that we collected from organisms with annotated growth temperatures. Our method, TemStaPro (Temperatures of Stability for Proteins), was used to predict thermostability of CRISPR-Cas Class II effector proteins (C2EPs). Predictions indicated sharp differences among groups of C2EPs in terms of thermostability and were largely in tune with previously published and our newly obtained experimental data. AVAILABILITY AND IMPLEMENTATION: TemStaPro software and the related data are freely available from https://github.com/ievapudz/TemStaPro and https://doi.org/10.5281/zenodo.7743637.

A new family of CRISPR-type V nucleases with C-rich PAM recognition.

Most CRISPR-type V nucleases are stimulated to cleave double-stranded (ds) DNA targets by a T-rich PAM, which restricts their targeting range. Here, we identify and characterize a new family of type V RNA-guided nuclease, Cas12l, that exclusively recognizes a C-rich (5'-CCY-3') PAM. The organization of genes within its CRISPR locus is similar to type II-B CRISPR-Cas9 systems, but both sequence analysis and functional studies establish it as a new family of type V effector. Biochemical experiments show that Cas12l nucleases function optimally between 37 and 52°C, depending on the ortholog, and preferentially cut supercoiled DNA. Like other type V nucleases, it exhibits collateral nonspecific ssDNA and ssRNA cleavage activity that is triggered by ssDNA or dsDNA target recognition. Finally, we show that one family member, Asp2Cas12l, functions in a heterologous cellular environment, altogether, suggesting that this new group of CRISPR-associated nucleases may be harnessed as genome editing reagents.

Rewiring Cas9 to Target New PAM Sequences.

Papers by Anders et al. (2016) and Hirano et al. (2016b), published in this issue of Molecular Cell, show that SpCas9 uses an induced fit mechanism to recognize altered protospacer adjacent motif (PAM) sequences.

5' modifications to CRISPR-Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage.

A key aim in exploiting CRISPR-Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9-gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3' end. In contrast, we found that 5' additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5' end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design.

DnaQ exonuclease-like domain of Cas2 promotes spacer integration in a type I-E CRISPR-Cas system.

CRISPR-Cas systems constitute an adaptive immune system that provides acquired resistance against phages and plasmids in prokaryotes. Upon invasion of foreign nucleic acids, some cells integrate short fragments of foreign DNA as spacers into the CRISPR locus to memorize the invaders and acquire resistance in the subsequent round of infection. This immunization step called adaptation is the least understood part of the CRISPR-Cas immunity. We have focused here on the adaptation stage of Streptococcus thermophilus DGCC7710 type I-E CRISPR4-Cas (St4) system. Cas1 and Cas2 proteins conserved in nearly all CRISPR-Cas systems are required for spacer acquisition. The St4 CRISPR-Cas system is unique because the Cas2 protein is fused to an additional DnaQ exonuclease domain. Here, we demonstrate that St4 Cas1 and Cas2-DnaQ form a multimeric complex, which is capable of integrating DNA duplexes with 3'-overhangs (protospacers) in vitro We further show that the DnaQ domain of Cas2 functions as a 3'-5'-exonuclease that processes 3'-overhangs of the protospacer to promote integration.

Genomic Characterization of Cyanophage vB_AphaS-CL131 Infecting Filamentous Diazotrophic Cyanobacterium Aphanizomenon flos-aquae Reveals Novel Insights into Virus-Bacterium Interactions.

While filamentous cyanobacteria play a crucial role in food web dynamics and biogeochemical cycling of many aquatic ecosystems around the globe, the knowledge regarding the phages infecting them is limited. Here, we describe the complete genome of the virulent cyanophage vB_AphaS-CL131 (here, CL 131), a Siphoviridae phage that infects the filamentous diazotrophic bloom-forming cyanobacterium Aphanizomenon flos-aquae in the brackish Baltic Sea. CL 131 features a 112,793-bp double-stranded DNA (dsDNA) genome encompassing 149 putative open reading frames (ORFs), of which the majority (86%) lack sequence homology to genes with known functions in other bacteriophages or bacteria. Phylogenetic analysis revealed that CL 131 possibly represents a new evolutionary lineage within the group of cyanophages infecting filamentous cyanobacteria, which form a separate cluster from phages infecting unicellular cyanobacteria. CL 131 encodes a putative type V-U2 CRISPR-Cas system with one spacer (out of 10) targeting a DNA primase pseudogene in a cyanobacterium and a putative type II toxin-antitoxin system, consisting of a GNAT family N-acetyltransferase and a protein of unknown function containing the PRK09726 domain (characteristic of HipB antitoxins). Comparison of CL 131 proteins to reads from Baltic Sea and other available fresh- and brackish-water metagenomes and analysis of CRISPR-Cas arrays in publicly available A. flos-aquae genomes demonstrated that phages similar to CL 131 are present and dynamic in the Baltic Sea and share a common history with their hosts dating back at least several decades. In addition, different CRISPR-Cas systems within individual A. flos-aquae genomes targeted several sequences in the CL 131 genome, including genes related to virion structure and morphogenesis. Altogether, these findings revealed new genomic information for exploring viral diversity and provide a model system for investigation of virus-host interactions in filamentous cyanobacteria.IMPORTANCE The genomic characterization of novel cyanophage vB_AphaS-CL131 and the analysis of its genomic features in the context of other viruses, metagenomic data, and host CRISPR-Cas systems contribute toward a better understanding of aquatic viral diversity and distribution in general and of brackish-water cyanophages infecting filamentous diazotrophic cyanobacteria in the Baltic Sea in particular. The results of this study revealed previously undescribed features of cyanophage genomes (e.g., self-excising intein-containing putative dCTP deaminase and putative cyanophage-encoded CRISPR-Cas and toxin-antitoxin systems) and can therefore be used to predict potential interactions between bloom-forming cyanobacteria and their cyanophages.

Methods for decoding Cas9 protospacer adjacent motif (PAM) sequences: A brief overview.

Recently the Cas9, an RNA guided DNA endonuclease, emerged as a powerful tool for targeted genome manipulations. Cas9 protein can be reprogrammed to cleave, bind or nick any DNA target by simply changing crRNA sequence, however a short nucleotide sequence, termed PAM, is required to initiate crRNA hybridization to the DNA target. PAM sequence is recognized by Cas9 protein and must be determined experimentally for each Cas9 variant. Exploration of Cas9 orthologs could offer a diversity of PAM sequences and novel biochemical properties that may be beneficial for genome editing applications. Here we briefly review and compare Cas9 PAM identification assays that can be adopted for other PAM-dependent CRISPR-Cas systems.

In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus.

Clustered regularly interspaced short palindromic repeats (CRISPR)-encoded immunity in Type I systems relies on the Cascade (CRISPR-associated complex for antiviral defence) ribonucleoprotein complex, which triggers foreign DNA degradation by an accessory Cas3 protein. To establish the mechanism for adaptive immunity provided by the Streptococcus thermophilus CRISPR4-Cas (CRISPR-associated) system (St-CRISPR4-Cas), we isolated an effector complex (St-Cascade) containing 61-nucleotide CRISPR RNA (crRNA). We show that St-Cascade, guided by crRNA, binds in vitro to a matching proto-spacer if a proto-spacer adjacent motif (PAM) is present. Surprisingly, the PAM sequence determined from binding analysis is promiscuous and limited to a single nucleotide (A or T) immediately upstream (-1 position) of the proto-spacer. In the presence of a correct PAM, St-Cascade binding to the target DNA generates an R-loop that serves as a landing site for the Cas3 ATPase/nuclease. We show that Cas3 binding to the displaced strand in the R-loop triggers DNA cleavage, and if ATP is present, Cas3 further degrades DNA in a unidirectional manner. These findings establish a molecular basis for CRISPR immunity in St-CRISPR4-Cas and other Type I systems.

Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome.

RNA-guided nucleases (RGNs) based on the type II CRISPR-Cas9 system of Streptococcus pyogenes (Sp) have been widely used for genome editing in experimental models. However, the nontrivial level of off-target activity reported in several human cells may hamper clinical translation. RGN specificity depends on both the guide RNA (gRNA) and the protospacer adjacent motif (PAM) recognized by the Cas9 protein. We hypothesized that more stringent PAM requirements reduce the occurrence of off-target mutagenesis. To test this postulation, we generated RGNs based on two Streptococcus thermophilus (St) Cas9 proteins, which recognize longer PAMs, and performed a side-by-side comparison of the three RGN systems targeted to matching sites in two endogenous human loci, PRKDC and CARD11. Our results demonstrate that in samples with comparable on-target cleavage activities, significantly lower off-target mutagenesis was detected using St-based RGNs as compared to the standard Sp-RGNs. Moreover, similarly to SpCas9, the StCas9 proteins accepted truncated gRNAs, suggesting that the specificities of St-based RGNs can be further improved. In conclusion, our results show that Cas9 proteins with longer or more restrictive PAM requirements provide a safe alternative to SpCas9-based RGNs and hence a valuable option for future human gene therapy applications.

Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes.

Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems protect bacteria and archaea from infection by viruses and plasmids. Central to this defense is a ribonucleoprotein complex that produces RNA-guided cleavage of foreign nucleic acids. In DNA-targeting CRISPR-Cas systems, the RNA component of the complex encodes target recognition by forming a site-specific hybrid (R-loop) with its complement (protospacer) on an invading DNA while displacing the noncomplementary strand. Subsequently, the R-loop structure triggers DNA degradation. Although these reactions have been reconstituted, the exact mechanism of R-loop formation has not been fully resolved. Here, we use single-molecule DNA supercoiling to directly observe and quantify the dynamics of torque-dependent R-loop formation and dissociation for both Cascade- and Cas9-based CRISPR-Cas systems. We find that the protospacer adjacent motif (PAM) affects primarily the R-loop association rates, whereas protospacer elements distal to the PAM affect primarily R-loop stability. Furthermore, Cascade has higher torque stability than Cas9 by using a conformational locking step. Our data provide direct evidence for directional R-loop formation, starting from PAM recognition and expanding toward the distal protospacer end. Moreover, we introduce DNA supercoiling as a quantitative tool to explore the sequence requirements and promiscuities of orthogonal CRISPR-Cas systems in rapidly emerging gene-targeting applications.

Design of a CRISPR-Cas system to increase resistance of Bacillus subtilis to bacteriophage SPP1.

Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (cas) genes form an adaptive prokaryotic immune system which provides acquired resistance against viruses and plasmids. Bacillus subtilis presently is the best-characterized laboratory model for Gram-positive bacteria and also widely used for industrial production of enzymes, vitamins and antibiotics. In this study, we show that type II-A CRISPR-Cas system from Streptococcus thermophilus can be transferred into B. subtilis and provides heterologous protection against phage infection. We engineered a heterologous host by cloning S. thermophilus Cas9 and a spacer targeting bacteriophage SPP1 into the chromosome of B. subtilis, which does not harbor its own CRISPR-Cas systems. We found that the heterologous CRISPR-Cas system is functionally active in B. subtilis and provides resistance against bacteriophage SPP1 infection. The high efficiency of the acquired immunity against phage could be useful in generation of biotechnologically important B. subtilis strains with engineered chromosomes.

Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.

Molecular mechanisms of CRISPR-mediated microbial immunity.

Bacteriophages (phages) infect bacteria in order to replicate and burst out of the host, killing the cell, when reproduction is completed. Thus, from a bacterial perspective, phages pose a persistent lethal threat to bacterial populations. Not surprisingly, bacteria evolved multiple defense barriers to interfere with nearly every step of phage life cycles. Phages respond to this selection pressure by counter-evolving their genomes to evade bacterial resistance. The antagonistic interaction between bacteria and rapidly diversifying viruses promotes the evolution and dissemination of bacteriophage-resistance mechanisms in bacteria. Recently, an adaptive microbial immune system, named clustered regularly interspaced short palindromic repeats (CRISPR) and which provides acquired immunity against viruses and plasmids, has been identified. Unlike the restriction­modification anti-phage barrier that subjects to cleavage any foreign DNA lacking a protective methyl-tag in the target site, the CRISPR­Cas systems are invader-specific, adaptive, and heritable. In this review, we focus on the molecular mechanisms of interference/immunity provided by different CRISPR­Cas systems.

Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.

Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate site-specific nicks on opposite DNA strands. Results demonstrate that the Cas9-crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and protein-mediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.

Programmable DNA cleavage in vitro by Cas9.

The ternary Cas9-crRNA-tracrRNA complex (Cas9t) of the Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system functions as an Mg2+-dependent RNA-directed DNA endonuclease that locates its DNA target guided by the crRNA (CRISPR RNA) in the tracrRNA-crRNA structure and introduces a double-strand break at a specific site in DNA. The simple modular organization of Cas9t, where specificity for the DNA target is encoded by a small crRNA and the cleavage reaction is executed by the Cas9 endonuclease, provides a versatile platform for the engineering of universal RNA-directed DNA endonucleases. By altering the crRNA sequence within the Cas9t complex, programmable endonucleases can be designed for both in vitro and in vivo applications. Cas9t has been recently employed as a gene-editing tool in various eukaryotic cell types. Using Streptococcus thermophilus Cas9t as a model system, we demonstrate the feasibility of Cas9t as a programmable molecular tool for in vitro DNA manipulations.

crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus.

The Cas9-crRNA complex of the Streptococcus thermophilus DGCC7710 CRISPR3-Cas system functions as an RNA-guided endonuclease with crRNA-directed target sequence recognition and protein-mediated DNA cleavage. We show here that an additional RNA molecule, tracrRNA (trans-activating CRISPR RNA), co-purifies with the Cas9 protein isolated from the heterologous E. coli strain carrying the S. thermophilus DGCC7710 CRISPR3-Cas system. We provide experimental evidence that tracrRNA is required for Cas9-mediated DNA interference both in vitro and in vivo. We show that Cas9 specifically promotes duplex formation between the precursor crRNA (pre-crRNA) transcript and tracrRNA, in vitro. Furthermore, the housekeeping RNase III contributes to primary pre-crRNA-tracrRNA duplex cleavage for mature crRNA biogenesis. RNase III, however, is not required in the processing of a short pre-crRNA transcribed from a minimal CRISPR array containing a single spacer. Finally, we show that an in vitro-assembled ternary Cas9-crRNA-tracrRNA complex cleaves DNA. This study further specifies the molecular basis for crRNA-based re-programming of Cas9 to specifically cleave any target DNA sequence for precise genome surgery. The processes for crRNA maturation and effector complex assembly established here will contribute to the further development of the Cas9 re-programmable system for genome editing applications.

The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.

The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

Diversification of the CRISPR Toolbox: Applications of CRISPR-Cas Systems Beyond Genome Editing.

The discovery of CRISPR has revolutionized the field of genome engineering, but the potential of this technology is far from reaching its limits. In this review, we explore the broad range of applications of CRISPR technology to highlight the rapid expansion of the field beyond gene editing alone. It has been demonstrated that CRISPR technology can control gene expression, spatiotemporally image the genome in vivo, and detect specific nucleic acid sequences for diagnostics. In addition, new technologies are under development to improve CRISPR quality controls for gene editing, thereby improving the reliability of these technologies for therapeutics and beyond. These are just some of the many CRISPR tools that have been developed in recent years, and the toolbox continues to diversify.