Diversity of CRISPR-Cas-mediated mechanisms of adaptive immunity in prokaryotes and their application in biotechnology

CRISPR-Cas systems of adaptive immunity in prokaryotes consist of CRISPR arrays (clusters of short repeated genomic DNA fragments separated by unique spacer sequences) and cas (CRISPR-associated) genes that provide cells with resistance against bacteriophages and plasmids containing protospacers, i.e. sequences complementary to CRISPR array spacers. CRISPR-Cas systems are responsible for two different cellular phenomena: CRISPR adaptation and CRISPR interference. CRISPR adaptation is cell genome modification by integration of new spacers that represents a unique case of Lamarckian inheritance. CRISPR interference involves specific recognition of protospacers in foreign DNA followed by introduction of breaks into this DNA and its destruction. According to the mechanisms of action, CRISPR-Cas systems have been subdivided into two classes, five types, and numerous subtypes. The development of techniques based on CRISPR interference mediated by the Type II system Cas9 protein has revolutionized the field of genome editing because it allows selective, efficient, and relatively simple introduction of directed breaks into target DNA loci. However, practical applications of CRISPR-Cas systems are not limited only to genome editing. In this review, we focus on the variety of CRISPR interference and CRISPR adaptation mechanisms and their prospective use in biotechnology.     

Diverse Mechanisms of CRISPR-Cas9 Inhibition by Type II Anti-CRISPR Proteins

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) systems provide bacteria and archaea with an adaptive immune response against invasion by mobile genetic elements like phages, plasmids, and transposons. These systems have been repurposed as very powerful biotechnological tools for gene editing applications in both bacterial and eukaryotic systems. The discovery of natural off-switches for CRISPR-Cas systems, known as anti-CRISPR proteins, provided a mechanism for controlling CRISPR-Cas activity and opened avenues for the development of more precise editing tools. In this review, we focus on the inhibitory mechanisms of anti-CRISPRs that are active against type II CRISPR-Cas systems and briefly discuss their biotechnological applications.     

CRISPR-Cas系统转录调控机制的研究进展

原核生物可利用由CRISPR-Cas系统(clustered regularly interspaced short palindromic repeats-CRISPR associated)介导的适应性免疫机制防御外源核酸入侵.在适应性免疫过程中,原核生物将外源核酸部分片段整合至自身CRISPR阵列中,表达并加工的...     

Cleavage of Phage DNA by the Streptococcus thermophilus CRISPR3-Cas System

Streptococcus thermophilus, similar to other Bacteria and Archaea, has developed defense mechanisms to protect cells against invasion by foreign nucleic acids, such as virus infections and plasmid transformations. One defense system recently described in these organisms is the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats loci coupled to CRISPR-associated genes). Two S. thermophilus CRISPR-Cas systems, CRISPR1-Cas and CRISPR3-Cas, have been shown to actively block phage infection. The CRISPR1-Cas system interferes by cleaving foreign dsDNA entering the cell in a length-specific and orientation-dependant manner. Here, we show that the S. thermophilus CRISPR3-Cas system acts by cleaving phage dsDNA genomes at the same specific position inside the targeted protospacer as observed with the CRISPR1-Cas system. Only one cleavage site was observed in all tested strains. Moreover, we observed that the CRISPR1-Cas and CRISPR3-Cas systems are compatible and, when both systems are present within the same cell, provide increased resistance against phage infection by both cleaving the invading dsDNA. We also determined that overall phage resistance efficiency is correlated to the total number of newly acquired spacers in both CRISPR loci.     

CRISPR-Cas systems target a diverse collection of invasive mobile genetic elements in human microbiomes

Background

Bacteria and archaea develop immunity against invading genomes by incorporating pieces of the invaders'' sequences, called spacers, into a clustered regularly interspaced short palindromic repeats (CRISPR) locus between repeats, forming arrays of repeat-spacer units. When spacers are expressed, they direct CRISPR-associated (Cas) proteins to silence complementary invading DNA. In order to characterize the invaders of human microbiomes, we use spacers from CRISPR arrays that we had previously assembled from shotgun metagenomic datasets, and identify contigs that contain these spacers'' targets.

Results

We discover 95,000 contigs that are putative invasive mobile genetic elements, some targeted by hundreds of CRISPR spacers. We find that oral sites in healthy human populations have a much greater variety of mobile genetic elements than stool samples. Mobile genetic elements carry genes encoding diverse functions: only 7% of the mobile genetic elements are similar to known phages or plasmids, although a much greater proportion contain phage- or plasmid-related genes. A small number of contigs share similarity with known integrative and conjugative elements, providing the first examples of CRISPR defenses against this class of element. We provide detailed analyses of a few large mobile genetic elements of various types, and a relative abundance analysis of mobile genetic elements and putative hosts, exploring the dynamic activities of mobile genetic elements in human microbiomes. A joint analysis of mobile genetic elements and CRISPRs shows that protospacer-adjacent motifs drive their interaction network; however, some CRISPR-Cas systems target mobile genetic elements lacking motifs.

Conclusions

We identify a large collection of invasive mobile genetic elements in human microbiomes, an important resource for further study of the interaction between the CRISPR-Cas immune system and invaders.     

微生物新型防御系统的系统性发现与展望

微生物防御系统是生物技术创新与发展的重要工具库,细菌限制性内切酶的发现催生了现代分子克隆技术,CRISPR系统的开发利用则使基因组编辑技术取得革命性突破。基于上述原因,微生物新型防御系统的发现与研究已引起各国科学家的重视,一些新的防御系统如pAgos和DISARM等相继被发现和研究。为进一步挖掘微生物中可能蕴藏着的其他未知防御系统,最近以色列科学家Sorek等报道了从海量的微生物基因组序列中系统性发现新型防御系统的研究策略,并且通过合成生物学思路鉴定了10种新型系统的抗病毒或抗质粒功能。本文将首先介绍Sorek团队系统性发现新型防御系统的研究工作,进而总结目前已知的主要微生物新型防御系统的可能机制,并对该领域的发展态势与挑战进行分析和展望。     

CRISPR-Cas系统调控细菌生理功能的机制研究进展

CRISPR-Cas系统是存在于部分细菌和绝大部分古细菌中的一种获得性免疫防御系统,使细菌在外源性基因入侵时具有免疫防御能力。此外,CRISPR-Cas系统对细菌自身生物膜的形成、耐药性、毒力等生理功能都有调控作用,这对于研究人员进行相关研究有着重要意义。本文以细菌CRISPR-Cas系统及其发挥免疫防御作用的相关研究为基础展开论述,重点阐述该系统对细菌生理功能的调控作用,并对其应用前景进行了展望,以期为进一步研究细菌耐药性和致病性提供新思路。     

On the global CRISPR array behavior in class I systems

Background

Much effort is underway to build and upgrade databases and tools related to occurrence, diversity, and characterization of CRISPR-Cas systems. As microbial communities and their genome complements are unearthed, much emphasis has been placed on details of individual strains and model systems within the CRISPR-Cas classification, and that collection of information as a whole affords the opportunity to analyze CRISPR-Cas systems from a quantitative perspective to gain insight into distribution of CRISPR array sizes across the different classes, types and subtypes. CRISPR diversity, nomenclature, occurrence, and biological functions have generated a plethora of data that created a need to understand the size and distribution of these various systems to appreciate their features and complexity.

Results

By utilizing a statistical framework and visual analytic techniques, we have been able to test several hypotheses about CRISPR loci in bacterial class I systems. Quantitatively, though CRISPR loci can expand to hundreds of spacers, the mean and median sizes are 40 and 25, respectively, reflecting rather modest acquisition and/or retention overall. Histograms uncovered that CRISPR array size displayed a parametric distribution, which was confirmed by a goodness-of fit test. Mapping the frequency of CRISPR loci on a standardized chromosome plot revealed that CRISPRs have a higher probability of occurring at clustered locations along the positive or negative strand. Lastly, when multiple arrays occur in a particular system, the size of a particular CRISPR array varies with its distance from the cas operon, reflecting acquisition and expansion biases.

Conclusions

This study establishes that bacterial Class I CRISPR array size tends to follow a geometric distribution; these CRISPRs are not randomly distributed along the chromosome; and the CRISPR array closest to the cas genes is typically larger than loci in trans. Overall, we provide an analytical framework to understand the features and behavior of CRISPR-Cas systems through a quantitative lens.

Reviewers

This article was reviewed by Eugene Koonin (NIH-NCBI) and Uri Gophna (Tel Aviv University).

     

Design of a CRISPR-Cas system to increase resistance of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> 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.     

Structural and mechanistic insights into the inhibition of type I-F CRISPR-Cas system by anti-CRISPR protein AcrIF23

Prokaryotes evolved clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins as a kind of adaptive immune defense against mobile genetic elements including harmful phages. To counteract this defense, many mobile genetic elements in turn encode anti-CRISPR proteins (Acrs) to inactivate the CRISPR-Cas system. While multiple mechanisms of Acrs have been uncovered, it remains unknown whether other mechanisms are utilized by uncharacterized Acrs. Here, we report a novel mechanism adopted by recently identified AcrIF23. We show that AcrIF23 interacts with the Cas2/3 helicase-nuclease in the type I-F CRISPR-Cas system, similar to AcrIF3. The structure of AcrIF23 demonstrated a novel fold and structure-based mutagenesis identified a surface region of AcrIF23 involved in both Cas2/3-binding and its inhibition capacity. Unlike AcrIF3, however, we found AcrIF23 only potently inhibits the DNA cleavage activity of Cas2/3 but does not hinder the recruitment of Cas2/3 to the CRISPR RNA-guided surveillance complex (the Csy complex). Also, in contrast to AcrIF3 which hinders substrate DNA recognition by Cas2/3, we show AcrIF23 promotes DNA binding to Cas2/3. Taken together, our study identifies a novel anti-CRISPR mechanism used by AcrIF23 and highlights the diverse mechanisms adopted by Acrs.     

COVID-19 one year later: a retrospect of CRISPR-Cas system in combating COVID-19

Coronavirus disease 2019 (COVID-19), an infectious disease caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a persistent global threat. The transmission of SARS-CoV-2 is wide and swift. Rapid detection of the viral RNA and effective therapy are imperative to prevent the worldwide spread of the new infectious disease. Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)- CRISPR-associated protein (Cas) system is an RNA-directed adaptive immune system, and it has been transformed into a gene editing tool. Applications of CRISPR-Cas system involves in many fields, such as human gene therapy, drug discovery and disease diagnosis. Under the background of COVID-19 pandemic, CRISPR-Cas system shows hidden capacity to fight the emergency in many aspects. This review will focus on the role of gene editing in COVID-19 diagnosis and treatment. We will describe the potential use of CRISPR-Cas-based system in combating COVID-19, from diagnosis to treatment. Furthermore, the limitation and perspectives of this novel technology are also evaluated.     

CRISPR-Cas系统及anti-CRISPR蛋白在微生物中的研究进展

CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISP) and CRISPR associated proteins)系统是细菌用来防御病毒、质粒等外源核酸入侵的一种获得性免疫防御系统。随着研究的深入,CRISPR-Cas系统已发展为一种重要的基因编辑工具,并成功应用于动物、植物和微生物的基因改造中。但该基因编辑方法有时存在基因脱靶效应,从而限制了其推广应用。最近,通过将1种新发现的抗CRISPR蛋白(Anti-CRISPR protein,ACP)与CRISPR-Cas系统相结合,已成功开发出可控制基因脱靶效率的CRISPR-Cas基因编辑工具。本文首先对CRISPR-Cas系统及ACP进行了简要介绍,然后就CRISPR-Cas基因编辑工具及ACP在微生物基因改造的应用现状进行了综述,并对ACP介导的CRISPR-Cas基因编辑方法(ACP-CRISPR-Cas)在微生物基因编辑中的应用前景进行了讨论。     

A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action

Background  

All archaeal and many bacterial genomes contain Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR) and variable arrays of the CRISPR-associated (cas) genes that have been previously implicated in a novel form of DNA repair on the basis of comparative analysis of their protein product sequences. However, the proximity of CRISPR and cas genes strongly suggests that they have related functions which is hard to reconcile with the repair hypothesis.     

CRISPR Content Correlates with the Pathogenic Potential of Escherichia coli

Guide RNA molecules (crRNA) produced from clustered regularly interspaced short palindromic repeat (CRISPR) arrays, altogether with effector proteins (Cas) encoded by cognate cas (CRISPR associated) genes, mount an interference mechanism (CRISPR-Cas) that limits acquisition of foreign DNA in Bacteria and Archaea. The specificity of this action is provided by the repeat intervening spacer carried in the crRNA, which upon hybridization with complementary sequences enables their degradation by a Cas endonuclease. Moreover, CRISPR arrays are dynamic landscapes that may gain new spacers from infecting elements or lose them for example during genome replication. Thus, the spacer content of a strain determines the diversity of sequences that can be targeted by the corresponding CRISPR-Cas system reflecting its functionality. Most Escherichia coli strains possess either type I-E or I-F CRISPR-Cas systems. To evaluate their impact on the pathogenicity of the species, we inferred the pathotype and pathogenic potential of 126 strains of this and other closely related species and analyzed their repeat content. Our results revealed a negative correlation between the number of I-E CRISPR units in this system and the presence of pathogenicity traits: the median number of repeats was 2.5-fold higher for commensal isolates (with 29.5 units, range 0–53) than for pathogenic ones (12.0, range 0–42). Moreover, the higher the number of virulence factors within a strain, the lower the repeat content. Additionally, pathogenic strains of distinct ecological niches (i.e., intestinal or extraintestinal) differ in repeat counts. Altogether, these findings support an evolutionary connection between CRISPR and pathogenicity in E. coli.     

A Complex of Cas Proteins 5, 6, and 7 Is Required for the Biogenesis and Stability of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1–8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.     

Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering

Non-coding RNAs (crRNAs) produced from clustered regularly interspaced short palindromic repeats (CRISPR) loci and CRISPR-associated (Cas) proteins of the prokaryotic CRISPR-Cas systems form complexes that interfere with the spread of transmissible genetic elements through Cas-catalysed cleavage of foreign genetic material matching the guide crRNA sequences. The easily programmable targeting of nucleic acids enabled by these ribonucleoproteins has facilitated the implementation of CRISPR-based molecular biology tools for in vivo and in vitro modification of DNA and RNA targets. Despite the diversity of DNA-targeting Cas nucleases so far identified, native and engineered derivatives of the Streptococcus pyogenes SpCas9 are the most widely used for genome engineering, at least in part due to their catalytic robustness and the requirement of an exceptionally short motif (5′-NGG-3′ PAM) flanking the target sequence. However, the large size of the SpCas9 variants impairs the delivery of the tool to eukaryotic cells and smaller alternatives are desirable. Here, we identify in a metagenome a new CRISPR-Cas9 system associated with a smaller Cas9 protein (EHCas9) that targets DNA sequences flanked by 5′-NGG-3′ PAMs. We develop a simplified EHCas9 tool that specifically cleaves DNA targets and is functional for genome editing applications in prokaryotes and eukaryotic cells.