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1.  Harnessing CRISPR-Cas9 immunity for genetic engineering 
CRISPR-Cas encodes an adaptive immune system that defends prokaryotes against infectious viruses and plasmids. Immunity is mediated by Cas nucleases, which use small RNA guides (the crRNAs) to specify a cleavage site within the genome of invading nucleic acids. In type II CRISPR-Cas systems, the DNA-cleaving activity is performed by a single enzyme Cas9 guided by an RNA duplex. Using synthetic single RNA guides, Cas9 can be reprogrammed to create specific double-stranded DNA breaks in the genomes of a variety of organisms, ranging from human cells to bacteria, and thus constitutes a powerful tool for genetic engineering. Here we describe recent advancements in our understanding of type II CRISPR-Cas immunity and how these studies led to revolutionary genome editing applications.
PMCID: PMC4155128  PMID: 25048165
2.  Adapting to new threats: the generation of memory by CRISPR-Cas immune systems 
Molecular microbiology  2014;93(1):1-9.
Clustered, regularly interspaced, short palindromic repeats (CRISPR) loci and their associated genes (cas) confer bacteria and archaea with adaptive immunity against phages and other invading genetic elements. A fundamental requirement of any immune system is the ability to build a memory of past infections in order to deal more efficiently with recurrent infections. The adaptive feature of CRISPR-Cas immune systems relies on their ability to memorize DNA sequences of invading molecules and integrate them in between the repetitive sequences of the CRISPR array in the form of “spacers”. The transcription of a spacer generates a small antisense RNA that is used by RNA-guided Cas nucleases to cleave the invading nucleic acid in order to protect the cell from infection. The acquisition of new spacers allows the CRISPR-Cas immune system to rapidly adapt against new threats and is therefore termed “adaptation”. Recent studies have begun to elucidate the genetic requirements for adaptation and have demonstrated that rather than being a stochastic process, the selection of new spacers is influenced by several factors. We review here our current knowledge of the CRISPR adaptation mechanism.
PMCID: PMC4104294  PMID: 24806524
CRISPR/Cas systems; spacer acquisition; adaptation; bacteriophage; adaptive immunity; horizontal gene transfer
3.  Development of sequence-specific antimicrobials based on programmable CRISPR-Cas nucleases 
Nature biotechnology  2014;32(11):1146-1150.
Antibiotics target conserved bacterial cellular pathways or growth functions and therefore cannot selectively kill specific members of a complex microbial population. Here, we develop programmable, sequence-specific antimicrobials using the RNA-guided nuclease Cas91, 2 delivered by a bacteriophage. We show that Cas9 re-programmed to target virulence genes kills virulent, but not avirulent, Staphylococcus aureus. Re-programming the nuclease to target antibiotic resistance genes destroys staphylococcal plasmids that harbor antibiotic resistance genes3, 4 and immunizes avirulent staphylococci to prevent the spread of plasmid-borne resistance genes. We also demonstrate the approach in vivo, showing its efficacy against S. aureus in a mouse skin colonization model. This new technology creates opportunities to manipulate complex bacterial populations in a sequence-specific manner.
PMCID: PMC4317352  PMID: 25282355
4.  Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting 
Nature  2014;514(7524):633-637.
A fundamental feature of immune systems is the ability to distinguish pathogenic from self and commensal elements, and to attack the former but tolerate the latter1. Prokaryotic CRISPR-Cas immune systems defend against phage infection using Cas nucleases and small RNA guides that specify one or more target sites for cleavage of the viral genome2,3. Temperate phages are viruses that can integrate into the bacterial chromosome, and they can carry genes that provide a fitness advantage to the lysogenic host4,5. However, CRISPR-Cas targeting that relies strictly on DNA sequence recognition provides indiscriminate immunity to both lytic and lysogenic infection by temperate phages6—compromising the genetic stability of these potentially beneficial elements altogether. Here we show that the Staphylococcus epidermidis CRISPR-Cas system can prevent lytic infection but tolerate lysogenization by temperate phages. Conditional tolerance is achieved through transcription-dependent DNA targeting, and ensures that targeting is resumed upon induction of the prophage lytic cycle. Our results provide evidence for the functional divergence of CRISPR-Cas systems and highlight the importance of targeting mechanism diversity. In addition, they extend the concept of ‘tolerance to non-self’ to the prokaryotic branch of adaptive immunity.
PMCID: PMC4214910  PMID: 25174707
5.  CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity 
Molecular cell  2014;54(2):234-244.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing, and can be repurposed for numerous DNA targeting applications including transcriptional control.
PMCID: PMC4025954  PMID: 24766887
6.  Genetic Characterization of Antiplasmid Immunity through a Type III-A CRISPR-Cas System 
Journal of Bacteriology  2014;196(2):310-317.
Many prokaryotes possess an adaptive immune system encoded by clustered regularly interspaced short palindromic repeats (CRISPRs). CRISPR loci produce small guide RNAs (crRNAs) that, in conjunction with flanking CRISPR-associated (cas) genes, combat viruses and block plasmid transfer by an antisense targeting mechanism. CRISPR-Cas systems have been classified into three types (I to III) that employ distinct mechanisms of crRNA biogenesis and targeting. The type III-A system in Staphylococcus epidermidis RP62a blocks the transfer of staphylococcal conjugative plasmids and harbors nine cas-csm genes. Previous biochemical analysis indicated that Cas10, Csm2, Csm3, Csm4, and Csm5 form a crRNA-containing ribonucleoprotein complex; however, the roles of these genes toward antiplasmid targeting remain unknown. Here, we determined the cas-csm genes that are required for antiplasmid immunity and used genetic and biochemical analyses to investigate the functions of predicted motifs and domains within these genes. We found that many mutations affected immunity by impacting the formation of the Cas10-Csm complex or crRNA biogenesis. Surprisingly, mutations in the predicted nuclease domains of the members of the Cas10-Csm complex had no detectable effect on antiplasmid immunity or crRNA biogenesis. In contrast, the deletion of csm6 and mutations in the cas10 Palm polymerase domain prevented CRISPR immunity without affecting either complex formation or crRNA production, suggesting their involvement in target destruction. By delineating the genetic requirements of this system, our findings further contribute to the mechanistic understanding of type III CRISPR-Cas systems.
PMCID: PMC3911255  PMID: 24187086
7.  Mobile DNA: an evolving field 
Mobile DNA  2014;5:16.
During the Mobile Genetic Elements and Genome Evolution Keystone Symposium in March 2014, the Editors of Mobile DNA caught up with a panel of conference speakers to select key advances in the field, and hear their thoughts on where mobile DNA research is going.
PMCID: PMC4046146  PMID: 24904692
8.  CRISPR decoys 
RNA Biology  2013;10(5):694-699.
CRISPR loci consist of an array of short repeats separated by spacer sequences that match the genome of viruses and plasmids that infect prokaryotes. Transcription of the CRISPR array generates small antisense RNAs that mediate immunity against these invaders. In recent years, there has been a notable increase in the investigation of CRISPR immunity, but studies have been restricted to organisms in which genetic manipulations are possible. Therefore, there is a need for the development of simple genetic tools that facilitate the study of this important pathway. Here we describe the use of CRISPR decoys, plasmids containing a non-transcribed repeat-spacer unit that disrupt CRISPR immunity. We show that decoys abrogate immunity against conjugation in S. epidermidis to levels comparable to a CRISPR deletion mutant. This technique can be used to generate full or spacer-specific CRISPR knockdowns in organisms in which decoy plasmids can be introduced.
PMCID: PMC3737327  PMID: 23584158
CRISPR; conjugation; genetic interference; immunity; staphylococcus
9.  DNA targeting specificity of RNA-guided Cas9 nucleases 
Nature biotechnology  2013;31(9):827-832.
The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of singleguide RNAs (sgRNAs) to enable genome editing1–10. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
PMCID: PMC3969858  PMID: 23873081
10.  CRISPR-Cas Immunity against Phages: Its Effects on the Evolution and Survival of Bacterial Pathogens 
PLoS Pathogens  2013;9(12):e1003765.
PMCID: PMC3861508  PMID: 24348245
11.  Control of gene expression by CRISPR-Cas systems 
F1000Prime Reports  2013;5:47.
Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated cas (CRISPR-associated) genes provide adaptive immunity against viruses (phages) and other mobile genetic elements in bacteria and archaea. While most of the early work has largely been dominated by examples of CRISPR-Cas systems directing the cleavage of phage or plasmid DNA, recent studies have revealed a more complex landscape where CRISPR-Cas loci might be involved in gene regulation. In this review, we summarize the role of these loci in the regulation of gene expression as well as the recent development of synthetic gene regulation using engineered CRISPR-Cas systems.
PMCID: PMC3816762  PMID: 24273648
12.  Multiplex Genome Engineering Using CRISPR/Cas Systems 
Science (New York, N.Y.)  2013;339(6121):819-823.
Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
PMCID: PMC3795411  PMID: 23287718
13.  Dealing with the Evolutionary Downside of CRISPR Immunity: Bacteria and Beneficial Plasmids 
PLoS Genetics  2013;9(9):e1003844.
The immune systems that protect organisms from infectious agents invariably have a cost for the host. In bacteria and archaea CRISPR-Cas loci can serve as adaptive immune systems that protect these microbes from infectiously transmitted DNAs. When those DNAs are borne by lytic viruses (phages), this protection can provide a considerable advantage. CRISPR-Cas immunity can also prevent cells from acquiring plasmids and free DNA bearing genes that increase their fitness. Here, we use a combination of experiments and mathematical-computer simulation models to explore this downside of CRISPR-Cas immunity and its implications for the maintenance of CRISPR-Cas loci in microbial populations. We analyzed the conjugational transfer of the staphylococcal plasmid pG0400 into Staphylococcus epidermidis RP62a recipients that bear a CRISPR-Cas locus targeting this plasmid. Contrary to what is anticipated for lytic phages, which evade CRISPR by mutations in the target region, the evasion of CRISPR immunity by plasmids occurs at the level of the host through loss of functional CRISPR-Cas immunity. The results of our experiments and models indicate that more than 10−4 of the cells in CRISPR-Cas positive populations are defective or deleted for the CRISPR-Cas region and thereby able to receive and carry the plasmid. Most intriguingly, the loss of CRISPR function even by large deletions can have little or no fitness cost in vitro. These theoretical and experimental results can account for the considerable variation in the existence, number and function of CRISPR-Cas loci within and between bacterial species. We postulate that as a consequence of the opposing positive and negative selection for immunity, CRISPR-Cas systems are in a continuous state of flux. They are lost when they bear immunity to laterally transferred beneficial genes, re-acquired by horizontal gene transfer, and ascend in environments where phage are a major source of mortality.
Author Summary
In addition to the virtue of protecting archaea and bacteria from the ravages of lethal viruses (phage), the immunity generated by the CRISPR-Cas systems have an evolutionary downside; they can prevent the acquisition of genes and genetic elements required for the adaptation and even the survival of these microbes. Using mathematical models and experiments with Staphylococcus epidermidis and the staphylococcal conjugative plasmid pG0400, we explore how bacteria deal with this evolutionary downside of CRISPR-Cas immunity. Although there are mechanisms by which immune populations of bacteria can acquire essential plasmids without the loss of CRISPR-Cas immunity, the results of our conjugation and fitness cost experiments suggest the most likely mechanism is the deactivation and deletion of this region. These results provide an explanation for the considerable variation in the existence, number and function of CRISPR-Cas within and between species of microbes. Along with other observations our work also suggests that the CRISPR-Cas loci are in a continuous state of flux: acquired by horizontal gene transfer, ascend when populations are confronted with phage and are rapidly lost when infectiously transmitted genes and genetic elements are required for the adaptation and survival of the population.
PMCID: PMC3784566  PMID: 24086164
14.  CRISPR-assisted editing of bacterial genomes 
Nature biotechnology  2013;31(3):233-239.
The targeting of nucleases to specific DNA sequences facilitates genome editing. Recent work demonstrated that the CRISPR-associated (Cas) nuclease Cas9 can be targeted to sequences in vitro simply by modifying a short7 CRISPR RNA (crRNA) guide. Here we use this CRISPR-Cas system to introduce marker-free mutations in Streptococcus pneumoniae and Escherichia coli. The approach involves re-programming Cas9 by using a crRNA complementary to a target chromosomal locus and introducing a template DNA harboring a desired mutation and an altered crRNA recognition site for recombination with the target locus. We exhaustively analyze Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements. Alone or together with recombineering, CRISPR assisted editing induces recombination at the targeted locus and kills non-edited cells leading to a recovery of close to a 100% of edited cells. Multiple crRNA can be used to modify several loci simultaneously. Our results show that CRISPR-mediated genome editing only requires programming of the crRNA and template sequences and thus constitutes a useful tool for genetic engineering.
PMCID: PMC3748948  PMID: 23360965
15.  Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system 
Nucleic Acids Research  2013;41(15):7429-7437.
The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.
PMCID: PMC3753641  PMID: 23761437
16.  MICROBIOLOGY: Slicer for DNA 
Nature  2010;468(7320):45-46.
Many bacteria and archaea protect themselves from viruses and other invasive genomes through a genetic interference pathway. The small RNAs that guide this defence specify the direct cleavage of foreign DNA.
PMCID: PMC3045673  PMID: 21048757
17.  CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea 
Nature reviews. Genetics  2010;11(3):181-190.
Sequence-directed genetic interference pathways control gene expression and preserve genome integrity in all kingdoms of life. The importance of such pathways is highlighted by the extensive study of RNA interference (RNAi) and related processes in eukaryotes. In many bacteria and most archaea, clustered, regularly interspaced short palindromic repeats (CRISPRs) are involved in a more recently discovered interference pathway that protects cells from bacteriophages and conjugative plasmids. CRISPR sequences provide an adaptive, heritable record of past infections and express CRISPR RNAs — small RNAs that target invasive nucleic acids. Here, we review the mechanisms of CRISPR interference and its roles in microbial physiology and evolution. We also discuss potential applications of this novel interference pathway.
PMCID: PMC2928866  PMID: 20125085
18.  Self vs. non-self discrimination during CRISPR RNA-directed immunity 
Nature  2010;463(7280):568-571.
All immune systems must distinguish self from non-self to repel invaders without inducing autoimmunity. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci protect bacteria and archaea from invasion by phage and plasmid DNA through a genetic interference pathway1–9. CRISPR loci are present in ~ 40% and ~90% of sequenced bacterial and archaeal genomes respectively10 and evolve rapidly, acquiring new spacer sequences to adapt to highly dynamic viral populations1, 11–13. Immunity requires a sequence match between the invasive DNA and the spacers that lie between CRISPR repeats1–9. Each cluster is genetically linked to a subset of the cas (CRISPR-associated) genes14–16 that collectively encode >40 families of proteins involved in adaptation and interference. CRISPR loci encode small CRISPR RNAs (crRNAs) that contain a full spacer flanked by partial repeat sequences2, 17–19. CrRNA spacers are thought to identify targets by direct Watson-Crick pairing with invasive “protospacer” DNA2, 3, but how they avoid targeting the spacer DNA within the encoding CRISPR locus itself is unknown. Here we have defined the mechanism of CRISPR self/non-self discrimination. In Staphylococcus epidermidis, target/crRNA mismatches at specific positions outside of the spacer sequence license foreign DNA for interference, whereas extended pairing between crRNA and CRISPR DNA repeats prevents autoimmunity. Hence, this CRISPR system uses the base-pairing potential of crRNAs not only to specify a target but also to spare the bacterial chromosome from interference. Differential complementarity outside of the spacer sequence is a built-in feature of all CRISPR systems, suggesting that this mechanism is a broadly applicable solution to the self/non-self dilemma that confronts all immune pathways.
PMCID: PMC2813891  PMID: 20072129
19.  Invasive DNA, Chopped and in the CRISPR 
In this issue of Structure, Wiedenheft et al. describe the structure and activity of Cas1, the only protein associated with all CRISPR loci. Cas1 is a metal-dependent deoxyribonuclease, consistent with a role in the adaptation phase of CRISPR immunity against invading nucleic acids.
PMCID: PMC2711432  PMID: 19523896
20.  CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA 
Science (New York, N.Y.)  2008;322(5909):1843-1845.
Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced short palindromic repeats (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate of Staphylococcus epidermidis harbors a CRISPR spacer that matches the nickase gene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation in S. epidermidis. Insertion of a self-splicing intron into nickase blocks interference despite the reconstitution of the target sequence in the spliced mRNA, indicating that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.
PMCID: PMC2695655  PMID: 19095942
21.  Sortase C-Mediated Anchoring of BasI to the Cell Wall Envelope of Bacillus anthracis▿  
Journal of Bacteriology  2007;189(17):6425-6436.
Vegetative forms of Bacillus anthracis replicate in tissues of an infected host and precipitate lethal anthrax disease. Upon host death, bacilli form dormant spores that contaminate the environment, thereby gaining entry into new hosts where spores germinate and once again replicate as vegetative forms. We show here that sortase C, an enzyme that is required for the formation of infectious spores, anchors BasI polypeptide to the envelope of predivisional sporulating bacilli. BasI anchoring to the cell wall requires the active site cysteine of sortase C and an LPNTA motif sorting signal at the C-terminal end of the BasI precursor. The LPNTA motif of BasI is cleaved between the threonine (T) and the alanine (A) residue; the C-terminal carboxyl group of threonine is subsequently amide linked to the side chain amino group of diaminopimelic acid within the wall peptides of B. anthracis peptidoglycan.
PMCID: PMC1951891  PMID: 17586639
22.  Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria 
The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.
PMCID: PMC1393253  PMID: 16524923
23.  Bacillus anthracis Sortase A (SrtA) Anchors LPXTG Motif-Containing Surface Proteins to the Cell Wall Envelope 
Journal of Bacteriology  2005;187(13):4646-4655.
Cell wall-anchored surface proteins of gram-positive pathogens play important roles during the establishment of many infectious diseases, but the contributions of surface proteins to the pathogenesis of anthrax have not yet been revealed. Cell wall anchoring in Staphylococcus aureus occurs by a transpeptidation mechanism requiring surface proteins with C-terminal sorting signals as well as sortase enzymes. The genome sequence of Bacillus anthracis encodes three sortase genes and eleven surface proteins with different types of cell wall sorting signals. Purified B. anthracis sortase A cleaved peptides encompassing LPXTG motif-type sorting signals between the threonine (T) and the glycine (G) residues in vitro. Sortase A activity could be inhibited by thiol-reactive reagents, similar to staphylococcal sortases. B. anthracis parent strain Sterne 34F2, but not variants lacking the srtA gene, anchored the collagen-binding MSCRAMM (microbial surface components recognizing adhesive matrix molecules) BasC (BA5258/BAS4884) to the bacterial cell wall. These results suggest that B. anthracis SrtA anchors surface proteins bearing LPXTG motif sorting signals to the cell wall envelope of vegetative bacilli.
PMCID: PMC1151759  PMID: 15968076

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