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CRISPR-Cas Systems

CRISPR-Cas Systems


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In the context of the bacterial systems (not the gene editing tool), I was wondering what happens to the foreign DNA after the Cas proteins have created a new spacer.

It is really not clear to me, most of the documentation I have found focuses on the subsequent steps (expression and interference) and do not discuss the fate of said foreign DNA. For example, see this image from wikipedia https://en.wikipedia.org/wiki/CRISPR#/media/File:Crispr.png">dna microbiology crispr

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CRISPR-Cas genome surveillance: From basic biology to transformative technology

Air date: Wednesday, March 11, 2015, 3:00:00 PM
Time displayed is Eastern Time, Washington DC Local
Views: Total views: 1,173 (383 Live, 790 On-demand)
Category: WALS - Wednesday Afternoon Lectures
Runtime: 01:05:13
Description: Wednesday Afternoon Lecture Series

Dr. Doudna, who specializes in the study of RNA, will present a brief history of the bacterial RNA-guided CRISPR biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism. Using CRISPR-Cas (clustered regularly interspaced short palindromic repeats) technology provides the foundation for remarkable developments in modifying, regulating, or marking genomic loci in a wide variety of cells and organisms. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way to fundamental discoveries in biology with applications in all branches of biotechnology, and strategies for human therapeutics. Dr. Doudna will discuss recent findings regarding the molecular mechanism of Cas9 and its use for targeted cell-based therapies.

About the annual Margaret Pittman Lecture:
This annual lecture honors Dr. Margaret Pittman, NIH’s first female lab chief, who made significant contributions to microbiology and vaccine development, particularly in the areas of pertussis and tetanus, during her long career at the National Institute of Allergy and Infectious Diseases.


New CRISPR Class Expands Genetic Engineering Toolbox

Biomedical engineers at Duke University have used a previously unexplored CRISPR technology to accurately regulate and edit genomes in human cells.

With this new approach, the researchers hope to dramatically expand the CRISPR-based tools available to biomedical engineers, opening up a new and diverse frontier of genome engineering technologies.

In a study appearing on Sept. 23 in Nature Biotechnology, Charles Gersbach, the Rooney Family Associate Professor of Biomedical Engineering at Duke, and Adrian Oliver, a post-doctoral fellow in the Gersbach lab who led the project, describe how they successfully harnessed Class 1 CRISPR systems to turn target genes on and off and edit the epigenome in human cells for the first time.

CRISPR-Cas is a defense system in which bacteria use RNA molecules and CRISPR-associated (Cas) proteins to target and destroy the DNA of invading viruses. The discovery of this phenomenon and the repurposing of the molecular machinery set off a genome-editing revolution as researchers learned how to wield the tool to specifically target and edit DNA in human cells.

CRISPR-Cas9, the most commonly used genome editing tool today, is categorized as a Class 2 CRISPR system. Class 2 systems are less common in the bacterial world, but they are theoretically simpler to work with, as they rely on only one Cas protein to target and cleave DNA.

Class 1 systems are not so simple, relying on multiple proteins working together in a complex called Cascade (CRISPR-associated complex for antiviral defense) to target DNA. After binding, Cascade recruits a Cas3 protein that cuts the DNA.

"If you were to look at the individual CRISPR systems of all the bacteria in the world, nearly 90 percent are Class 1 systems," said Gersbach. "CRISPR-Cas biology is an incredible source for biotechnology tools, but until recently everyone has only been looking at a small slice of the pie."

To demonstrate the capabilities of the Class 1 system, Oliver attached gene activators to specific sites along a type I E. coli Cascade complex and targeted the system to bind gene promoters, which regulate gene expression levels. Because she did not include the Cas3 protein in the experiment, there was no cutting of the DNA and no change to underlying DNA sequence. The experiment showed that the Cascade activator not only binds to the correct site and can turn up the levels of the target gene, but does so with accuracy and specificity comparable to CRISPR/Cas9.

Oliver repeated the process using type I Cascade complexes from an additional bacterial strain that was particularly robust in working at a variety of target sites. She also showed that the activator domain could be swapped for a repressor to turn target genes off. Again, the researchers noted accuracy and specificity comparable to CRISPR/Cas9 methods.

"We have found Cascade's structure to be remarkably modular, allowing for a variety of sites to attach activators or repressors, which are great tools for altering gene expression in human cells," Oliver said. "The flexible nature of Cascade makes it a promising genome engineering technology."

Gersbach and Oliver were encouraged to investigate the more complicated Class 1 CRISPR systems by their collaborators at nearby North Carolina State University, Professors Rodolphe Barrangou and Chase Beisel, who is now at the Helmholtz Centre for Infection Research in Germany. Barrangou is a microbiologist who has studied the natural biology of diverse CRISPR defense mechanisms for nearly two decades, and Beisel is a chemical engineer who has worked with Barrangou on engineering microorganisms with Class 1 CRISPR systems. They were both curious whether Gersbach's lab could use these systems in human cells similar to their work with Cas9.

"This work and the resulting technologies are a fantastic example of how collaboration across disciplines and across universities in the North Carolina Research Triangle can be highly innovative and productive" says Barrangou, the Todd R. Klaenhammer Distinguished Professor in Probiotics Research at North Carolina State University.

Now, the team is optimistic that their study, and the related work of others in the field, will incentivize new research into Class 1 CRISPR systems.

"The purpose of this project was to explore the diversity of CRISPR systems," said Gersbach. "There have been thousands of papers about CRISPR-Cas9 in the last decade, and yet we're constantly learning new things about it. With this study we're applying that mindset to the other 90% of what's out there."

So far, the team has shown that these Class 1 systems are comparable to to CRISPR-Cas9 in terms of accuracy and application. As they consider future directions, they are curious to explore how these systems differ from their Class 2 counterparts, and how these differences could prove useful for biotechnology applications.

The team is also interested in studying how Class 1 systems could address general challenges for CRISPR-Cas research, especially issues that complicate potential therapeutic applications, like immune responses to Cas proteins and concurrently using multiple types of CRISPR for different genome engineering functions.

"We know CRISPR could have a big impact on human health," said Gersbach. "But we're still at the very beginning of understanding how CRISPR is going to be used, what it can do, and what systems are available to us. We expect that this new tool will enable new areas of genome engineering."

This work was supported by the National Institutes of Health, the National Science Foundation, Locus Biosciences, an Allen Distinguished Investigator Award from the Paul G. Allen Frontiers Group, the Thorek Memorial Foundation, a Pfizer-NCBiotech Distinguished Postdoctoral Fellowship, and internal funds from Duke University and North Carolina State University.

Charles Gersbach, Adrian Pickar-Oliver, Rodolphe Barrangou, and Chase Beisel have filed patent applications related to genome engineering with Type I CRISPR systems. Charles Gersbach is a co-founder and advisor to Locus Biosciences and Element Genomics, and an advisor to Sarepta Therapeutics. Rodolphe Barrangou is a co-founder and Science Advisory Board member of Locus Biosciences and Intellia Therapeutics. Chase Beisel is a co-founder and Science Advisory Board member of Locus Biosciences.


Contents

Repeated sequences Edit

The discovery of clustered DNA repeats occurred independently in three parts of the world. The first description of what would later be called CRISPR is from Osaka University researcher Yoshizumi Ishino and his colleagues in 1987. They accidentally cloned part of a CRISPR sequence together with the "iap" gene (isozyme conversion of alkaline phosphatase) from the genome of Escherichia coli [14] [15] that was their target. The organization of the repeats was unusual. Repeated sequences are typically arranged consecutively, without interspersed different sequences. [15] [11] They did not know the function of the interrupted clustered repeats.

In 1993, researchers of Mycobacterium tuberculosis in the Netherlands published two articles about a cluster of interrupted direct repeats (DR) in that bacterium. They recognized the diversity of the sequences that intervened the direct repeats among different strains of M. tuberculosis [16] and used this property to design a typing method that was named spoligotyping, which is still in use today. [17] [18]

Francisco Mojica at the University of Alicante in Spain studied repeats observed in the archaeal organisms of Haloferax and Haloarcula species, and their function. Mojica's supervisor surmised at the time that the clustered repeats had a role in correctly segregating replicated DNA into daughter cells during cell division because plasmids and chromosomes with identical repeat arrays could not coexist in Haloferax volcanii. Transcription of the interrupted repeats was also noted for the first time this was the first full characterization of CRISPR. [18] [19] By 2000, Mojica performed a survey of scientific literature and one of his students performed a search in published genomes with a program devised by himself. They identified interrupted repeats in 20 species of microbes as belonging to the same family. [20] In 2001, Mojica and Ruud Jansen, who were searching for additional interrupted repeats, proposed the acronym CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to alleviate the confusion stemming from the numerous acronyms used to describe the sequences in the scientific literature. [19] [21] In 2002, Tang, et al. showed evidence that CRISPR repeat regions from the genome of Archaeoglobus fulgidus were transcribed into long RNA molecules that were subsequently processed into unit-length small RNAs, plus some longer forms of 2, 3, or more spacer-repeat units. [22] [23]

In 2005, yogurt researcher Rodolphe Barrangou, discovered that Streptococcus thermophilus, after iterative phage challenges, develops increased phage resistance, and this enhanced resistance is due to incorporation of additional CRISPR spacer sequences. [24] The Danish food company Danisco, which at that time Barrangou worked for, then developed phage resistant S. thermophilus strains for use in yogurt production. Danisco was later bought out by DuPont, which "owns about 50 percent of the global dairy culture market" and the technology went mainstream. [25]

CRISPR-associated systems Edit

A major addition to the understanding of CRISPR came with Jansen's observation that the prokaryote repeat cluster was accompanied by a set of homologous genes that make up CRISPR-associated systems or cas genes. Four cas genes (cas 1–4) were initially recognized. The Cas proteins showed helicase and nuclease motifs, suggesting a role in the dynamic structure of the CRISPR loci. [26] In this publication the acronym CRISPR was used as the universal name of this pattern. However, the CRISPR function remained enigmatic.

In 2005, three independent research groups showed that some CRISPR spacers are derived from phage DNA and extrachromosomal DNA such as plasmids. [30] [31] [32] In effect, the spacers are fragments of DNA gathered from viruses that previously tried to attack the cell. The source of the spacers was a sign that the CRISPR/cas system could have a role in adaptive immunity in bacteria. [27] [33] All three studies proposing this idea were initially rejected by high-profile journals, but eventually appeared in other journals. [34]

The first publication [31] proposing a role of CRISPR-Cas in microbial immunity, by Mojica and collaborators at the University of Alicante, predicted a role for the RNA transcript of spacers on target recognition in a mechanism that could be analogous to the RNA interference system used by eukaryotic cells. Koonin and colleagues extended this RNA interference hypothesis by proposing mechanisms of action for the different CRISPR-Cas subtypes according to the predicted function of their proteins. [35]

Experimental work by several groups revealed the basic mechanisms of CRISPR-Cas immunity. In 2007, the first experimental evidence that CRISPR was an adaptive immune system was published. [11] [4] A CRISPR region in Streptococcus thermophilus acquired spacers from the DNA of an infecting bacteriophage. The researchers manipulated the resistance of S. thermophilus to different types of phage by adding and deleting spacers whose sequence matched those found in the tested phages. [36] [37] In 2008, Brouns and Van der Oost identified a complex of Cas proteins (called Cascade) that in E. coli cut the CRISPR RNA precursor within the repeats into mature spacer-containing RNA molecules called CRISPR RNA (crRNA), which remained bound to the protein complex. [38] Moreover, it was found that Cascade, crRNA and a helicase/nuclease (Cas3) were required to provide a bacterial host with immunity against infection by a DNA virus. By designing an anti-virus CRISPR, they demonstrated that two orientations of the crRNA (sense/antisense) provided immunity, indicating that the crRNA guides were targeting dsDNA. That year Marraffini and Sontheimer confirmed that a CRISPR sequence of S. epidermidis targeted DNA and not RNA to prevent conjugation. This finding was at odds with the proposed RNA-interference-like mechanism of CRISPR-Cas immunity, although a CRISPR-Cas system that targets foreign RNA was later found in Pyrococcus furiosus. [11] [36] A 2010 study showed that CRISPR-Cas cuts both strands of phage and plasmid DNA in S. thermophilus. [39]

Cas9 Edit

Researchers studied a simpler CRISPR system from Streptococcus pyogenes that relies on the protein Cas9. The Cas9 endonuclease is a four-component system that includes two small molecules: crRNA and trans-activating CRISPR RNA (tracrRNA). [40] [41] Jennifer Doudna and Emmanuelle Charpentier re-engineered the Cas9 endonuclease into a more manageable two-component system by fusing the two RNA molecules into a "single-guide RNA" that, when combined with Cas9, could find and cut the DNA target specified by the guide RNA. This contribution was so significant that it was recognized by the Nobel Prize in Chemistry in 2020. By manipulating the nucleotide sequence of the guide RNA, the artificial Cas9 system could be programmed to target any DNA sequence for cleavage. [42] Another group of collaborators comprising Virginijus Šikšnys together with Gasiūnas, Barrangou and Horvath showed that Cas9 from the S. thermophilus CRISPR system can also be reprogrammed to target a site of their choosing by changing the sequence of its crRNA. These advances fueled efforts to edit genomes with the modified CRISPR-Cas9 system. [18]

Groups led by Feng Zhang and George Church simultaneously published descriptions of genome editing in human cell cultures using CRISPR-Cas9 for the first time. [11] [43] [44] It has since been used in a wide range of organisms, including baker's yeast (Saccharomyces cerevisiae), [45] [46] [47] the opportunistic pathogen Candida albicans, [48] [49] zebrafish (Danio rerio), [50] fruit flies (Drosophila melanogaster), [51] [52] ants (Harpegnathos saltator [53] and Ooceraea biroi [54] ), mosquitoes (Aedes aegypti [55] ), nematodes (Caenorhabditis elegans), [56] plants, [57] mice, [58] [59] monkeys [60] and human embryos. [61]

CRISPR has been modified to make programmable transcription factors that allow scientists to target and activate or silence specific genes. [62]

The CRISPR-Cas9 system has shown to make effective gene edits in Human tripronuclear zygotes first described in a 2015 paper by Chinese scientists P. Liang and Y. Xu. The system made a successful cleavage of mutant Beta-Hemoglobin (HBB) in 28 out of 54 embryos. 4 out of the 28 embryos were successfully recombined using a donor template given by the scientists. The scientists showed that during DNA recombination of the cleaved strand, the homologous endogenous sequence HBD competes with the exogenous donor template. DNA repair in human embryos is much more complicated and particular than in derived stem cells. [63]

Cas12a (formerly Cpf1) Edit

In 2015, the nuclease Cas12a (formerly known as Cpf1 [64] ) was characterized in the CRISPR/Cpf1 system of the bacterium Francisella novicida. [65] [66] Its original name, from a TIGRFAMs protein family definition built in 2012, reflects the prevalence of its CRISPR-Cas subtype in the Prevotella and Francisella lineages. Cas12a showed several key differences from Cas9 including: causing a 'staggered' cut in double stranded DNA as opposed to the 'blunt' cut produced by Cas9, relying on a 'T rich' PAM (providing alternative targeting sites to Cas9) and requiring only a CRISPR RNA (crRNA) for successful targeting. By contrast Cas9 requires both crRNA and a transactivating crRNA (tracrRNA).

These differences may give Cas12a some advantages over Cas9. For example, Cas12a's small crRNAs are ideal for multiplexed genome editing, as more of them can be packaged in one vector than can Cas9's sgRNAs. As well, the sticky 5′ overhangs left by Cas12a can be used for DNA assembly that is much more target-specific than traditional Restriction Enzyme cloning. [67] Finally, Cas12a cleaves DNA 18–23 base pairs downstream from the PAM site. This means there is no disruption to the recognition sequence after repair, and so Cas12a enables multiple rounds of DNA cleavage. By contrast, since Cas9 cuts only 3 base pairs upstream of the PAM site, the NHEJ pathway results in indel mutations that destroy the recognition sequence, thereby preventing further rounds of cutting. In theory, repeated rounds of DNA cleavage should cause an increased opportunity for the desired genomic editing to occur. [68] A distinctive feature of Cas12a, as compared to Cas9, is that after cutting its target, Cas12a remains bound to the target and then cleaves other ssDNA molecules non-discriminately. [69] This property is called "collateral cleavage" or "trans-cleavage" activity and has been exploited for the development of various diagnostic technologies. [70] [71]

Cas13 (formerly C2c2) Edit

In 2016, the nuclease Cas13a (formerly known as C2c2) from the bacterium Leptotrichia shahii was characterized. Cas13 is an RNA-guided RNA endonuclease, which means that it does not cleave DNA, but only single-stranded RNA. Cas13 is guided by its crRNA to a ssRNA target and binds and cleaves the target. Similar to Cas12a, the Cas13 remains bound to the target and then cleaves other ssRNA molecules non-discriminately. [72] This collateral cleavage property has been exploited for the development of various diagnostic technologies. [73] [74] [75]

Repeats and spacers Edit

The CRISPR array is made up of an AT-rich leader sequence followed by short repeats that are separated by unique spacers. [76] CRISPR repeats typically range in size from 28 to 37 base pairs (bps), though there can be as few as 23 bp and as many as 55 bp. [77] Some show dyad symmetry, implying the formation of a secondary structure such as a stem-loop ('hairpin') in the RNA, while others are designed to be unstructured. The size of spacers in different CRISPR arrays is typically 32 to 38 bp (range 21 to 72 bp). [77] New spacers can appear rapidly as part of the immune response to phage infection. [78] There are usually fewer than 50 units of the repeat-spacer sequence in a CRISPR array. [77]

CRISPR RNA structures Edit

Cas genes and CRISPR subtypes Edit

Small clusters of cas genes are often located next to CRISPR repeat-spacer arrays. Collectively the 93 cas genes are grouped into 35 families based on sequence similarity of the encoded proteins. 11 of the 35 families form the cas core, which includes the protein families Cas1 through Cas9. A complete CRISPR-Cas locus has at least one gene belonging to the cas core. [79]

CRISPR-Cas systems fall into two classes. Class 1 systems use a complex of multiple Cas proteins to degrade foreign nucleic acids. Class 2 systems use a single large Cas protein for the same purpose. Class 1 is divided into types I, III, and IV class 2 is divided into types II, V, and VI. [80] The 6 system types are divided into 19 subtypes. [81] Each type and most subtypes are characterized by a "signature gene" found almost exclusively in the category. Classification is also based on the complement of cas genes that are present. Most CRISPR-Cas systems have a Cas1 protein. The phylogeny of Cas1 proteins generally agrees with the classification system. [79] Many organisms contain multiple CRISPR-Cas systems suggesting that they are compatible and may share components. [82] [83] The sporadic distribution of the CRISPR/Cas subtypes suggests that the CRISPR/Cas system is subject to horizontal gene transfer during microbial evolution.

CRISPR-Cas immunity is a natural process of bacteria and archaea. [98] CRISPR-Cas prevents bacteriophage infection, conjugation and natural transformation by degrading foreign nucleic acids that enter the cell. [36]

Spacer acquisition Edit

When a microbe is invaded by a bacteriophage, the first stage of the immune response is to capture phage DNA and insert it into a CRISPR locus in the form of a spacer. Cas1 and Cas2 are found in both types of CRISPR-Cas immune systems, which indicates that they are involved in spacer acquisition. Mutation studies confirmed this hypothesis, showing that removal of cas1 or cas2 stopped spacer acquisition, without affecting CRISPR immune response. [99] [100] [101] [102] [103]

Multiple Cas1 proteins have been characterised and their structures resolved. [104] [105] [106] Cas1 proteins have diverse amino acid sequences. However, their crystal structures are similar and all purified Cas1 proteins are metal-dependent nucleases/integrases that bind to DNA in a sequence-independent manner. [82] Representative Cas2 proteins have been characterised and possess either (single strand) ssRNA- [107] or (double strand) dsDNA- [108] [109] specific endoribonuclease activity.

In the I-E system of E. coli Cas1 and Cas2 form a complex where a Cas2 dimer bridges two Cas1 dimers. [110] In this complex Cas2 performs a non-enzymatic scaffolding role, [110] binding double-stranded fragments of invading DNA, while Cas1 binds the single-stranded flanks of the DNA and catalyses their integration into CRISPR arrays. [111] [112] [113] New spacers are usually added at the beginning of the CRISPR next to the leader sequence creating a chronological record of viral infections. [114] In E. coli a histone like protein called integration host factor (IHF), which binds to the leader sequence, is responsible for the accuracy of this integration. [115] IHF also enhances integration efficiency in the type I-F system of Pectobacterium atrosepticum. [116] but in other systems different host factors may be required [117]

Protospacer adjacent motifs Edit

Bioinformatic analysis of regions of phage genomes that were excised as spacers (termed protospacers) revealed that they were not randomly selected but instead were found adjacent to short (3–5 bp) DNA sequences termed protospacer adjacent motifs (PAM). Analysis of CRISPR-Cas systems showed PAMs to be important for type I and type II, but not type III systems during acquisition. [32] [118] [119] [120] [121] [122] In type I and type II systems, protospacers are excised at positions adjacent to a PAM sequence, with the other end of the spacer cut using a ruler mechanism, thus maintaining the regularity of the spacer size in the CRISPR array. [123] [124] The conservation of the PAM sequence differs between CRISPR-Cas systems and appears to be evolutionarily linked to Cas1 and the leader sequence. [122] [125]

New spacers are added to a CRISPR array in a directional manner, [30] occurring preferentially, [78] [118] [119] [126] [127] but not exclusively, adjacent [121] [124] to the leader sequence. Analysis of the type I-E system from E. coli demonstrated that the first direct repeat adjacent to the leader sequence, is copied, with the newly acquired spacer inserted between the first and second direct repeats. [102] [123]

The PAM sequence appears to be important during spacer insertion in type I-E systems. That sequence contains a strongly conserved final nucleotide (nt) adjacent to the first nt of the protospacer. This nt becomes the final base in the first direct repeat. [103] [128] [129] This suggests that the spacer acquisition machinery generates single-stranded overhangs in the second-to-last position of the direct repeat and in the PAM during spacer insertion. However, not all CRISPR-Cas systems appear to share this mechanism as PAMs in other organisms do not show the same level of conservation in the final position. [125] It is likely that in those systems, a blunt end is generated at the very end of the direct repeat and the protospacer during acquisition.

Insertion variants Edit

Analysis of Sulfolobus solfataricus CRISPRs revealed further complexities to the canonical model of spacer insertion, as one of its six CRISPR loci inserted new spacers randomly throughout its CRISPR array, as opposed to inserting closest to the leader sequence. [124]

Multiple CRISPRs contain many spacers to the same phage. The mechanism that causes this phenomenon was discovered in the type I-E system of E. coli. A significant enhancement in spacer acquisition was detected where spacers already target the phage, even mismatches to the protospacer. This ‘priming’ requires the Cas proteins involved in both acquisition and interference to interact with each other. Newly acquired spacers that result from the priming mechanism are always found on the same strand as the priming spacer. [103] [128] [129] This observation led to the hypothesis that the acquisition machinery slides along the foreign DNA after priming to find a new protospacer. [129]

Biogenesis Edit

CRISPR-RNA (crRNA), which later guides the Cas nuclease to the target during the interference step, must be generated from the CRISPR sequence. The crRNA is initially transcribed as part of a single long transcript encompassing much of the CRISPR array. [28] This transcript is then cleaved by Cas proteins to form crRNAs. The mechanism to produce crRNAs differs among CRISPR/Cas systems. In type I-E and type I-F systems, the proteins Cas6e and Cas6f respectively, recognise stem-loops [130] [131] [132] created by the pairing of identical repeats that flank the crRNA. [133] These Cas proteins cleave the longer transcript at the edge of the paired region, leaving a single crRNA along with a small remnant of the paired repeat region.

Type III systems also use Cas6, however their repeats do not produce stem-loops. Cleavage instead occurs by the longer transcript wrapping around the Cas6 to allow cleavage just upstream of the repeat sequence. [134] [135] [136]

Type II systems lack the Cas6 gene and instead utilize RNaseIII for cleavage. Functional type II systems encode an extra small RNA that is complementary to the repeat sequence, known as a trans-activating crRNA (tracrRNA). [40] Transcription of the tracrRNA and the primary CRISPR transcript results in base pairing and the formation of dsRNA at the repeat sequence, which is subsequently targeted by RNaseIII to produce crRNAs. Unlike the other two systems the crRNA does not contain the full spacer, which is instead truncated at one end. [91]

CrRNAs associate with Cas proteins to form ribonucleotide complexes that recognize foreign nucleic acids. CrRNAs show no preference between the coding and non-coding strands, which is indicative of an RNA-guided DNA-targeting system. [5] [39] [99] [103] [137] [138] [139] The type I-E complex (commonly referred to as Cascade) requires five Cas proteins bound to a single crRNA. [140] [141]

Interference Edit

During the interference stage in type I systems the PAM sequence is recognized on the crRNA-complementary strand and is required along with crRNA annealing. In type I systems correct base pairing between the crRNA and the protospacer signals a conformational change in Cascade that recruits Cas3 for DNA degradation.

Type II systems rely on a single multifunctional protein, Cas9, for the interference step. [91] Cas9 requires both the crRNA and the tracrRNA to function and cleaves DNA using its dual HNH and RuvC/RNaseH-like endonuclease domains. Basepairing between the PAM and the phage genome is required in type II systems. However, the PAM is recognized on the same strand as the crRNA (the opposite strand to type I systems).

Type III systems, like type I require six or seven Cas proteins binding to crRNAs. [142] [143] The type III systems analysed from S. solfataricus and P. furiosus both target the mRNA of phages rather than phage DNA genome, [83] [143] which may make these systems uniquely capable of targeting RNA-based phage genomes. [82] Type III systems were also found to target DNA in addition to RNA using a different Cas protein in the complex, Cas10. [144] The DNA cleavage was shown to be transcription dependent. [145]

The mechanism for distinguishing self from foreign DNA during interference is built into the crRNAs and is therefore likely common to all three systems. Throughout the distinctive maturation process of each major type, all crRNAs contain a spacer sequence and some portion of the repeat at one or both ends. It is the partial repeat sequence that prevents the CRISPR-Cas system from targeting the chromosome as base pairing beyond the spacer sequence signals self and prevents DNA cleavage. [146] RNA-guided CRISPR enzymes are classified as type V restriction enzymes.

The cas genes in the adaptor and effector modules of the CRISPR-Cas system are believed to have evolved from two different ancestral modules. A transposon-like element called casposon encoding the Cas1-like integrase and potentially other components of the adaptation module was inserted next to the ancestral effector module, which likely functioned as an independent innate immune system. [147] The highly conserved cas1 and cas2 genes of the adaptor module evolved from the ancestral module while a variety of class 1 effector cas genes evolved from the ancestral effector module. [148] The evolution of these various class 1 effector module cas genes was guided by various mechanisms, such as duplication events. [149] On the other hand, each type of class 2 effector module arose from subsequent independent insertions of mobile genetic elements. [150] These mobile genetic elements took the place of the multiple gene effector modules to create single gene effector modules that produce large proteins which perform all the necessary tasks of the effector module. [150] The spacer regions of CRISPR-Cas systems are taken directly from foreign mobile genetic elements and thus their long term evolution is hard to trace. [151] The non-random evolution of these spacer regions has been found to be highly dependent on the environment and the particular foreign mobile genetic elements it contains. [152]

CRISPR/Cas can immunize bacteria against certain phages and thus halt transmission. For this reason, Koonin described CRISPR/Cas as a Lamarckian inheritance mechanism. [153] However, this was disputed by a critic who noted, "We should remember [Lamarck] for the good he contributed to science, not for things that resemble his theory only superficially. Indeed, thinking of CRISPR and other phenomena as Lamarckian only obscures the simple and elegant way evolution really works". [154] But as more recent studies have been conducted, it has become apparent that the acquired spacer regions of CRISPR-Cas systems are indeed a form of Lamarckian evolution because they are genetic mutations that are acquired and then passed on. [155] On the other hand, the evolution of the Cas gene machinery that facilitates the system evolves through classic Darwinian evolution. [155]

Coevolution Edit

Analysis of CRISPR sequences revealed coevolution of host and viral genomes. [156] Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR/Cas-mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during interaction with eukaryotic hosts. For example, Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence. [157]

The basic model of CRISPR evolution is newly incorporated spacers driving phages to mutate their genomes to avoid the bacterial immune response, creating diversity in both the phage and host populations. To resist a phage infection, the sequence of the CRISPR spacer must correspond perfectly to the sequence of the target phage gene. Phages can continue to infect their hosts given point mutations in the spacer. [146] Similar stringency is required in PAM or the bacterial strain remains phage sensitive. [119] [146]

Rates Edit

A study of 124 S. thermophilus strains showed that 26% of all spacers were unique and that different CRISPR loci showed different rates of spacer acquisition. [118] Some CRISPR loci evolve more rapidly than others, which allowed the strains' phylogenetic relationships to be determined. A comparative genomic analysis showed that E. coli and S. enterica evolve much more slowly than S. thermophilus. The latter's strains that diverged 250 thousand years ago still contained the same spacer complement. [158]

Metagenomic analysis of two acid-mine-drainage biofilms showed that one of the analyzed CRISPRs contained extensive deletions and spacer additions versus the other biofilm, suggesting a higher phage activity/prevalence in one community than the other. [78] In the oral cavity, a temporal study determined that 7–22% of spacers were shared over 17 months within an individual while less than 2% were shared across individuals. [127]

From the same environment a single strain was tracked using PCR primers specific to its CRISPR system. Broad-level results of spacer presence/absence showed significant diversity. However, this CRISPR added 3 spacers over 17 months, [127] suggesting that even in an environment with significant CRISPR diversity some loci evolve slowly.

CRISPRs were analysed from the metagenomes produced for the human microbiome project. [159] Although most were body-site specific, some within a body site are widely shared among individuals. One of these loci originated from streptococcal species and contained ≈15,000 spacers, 50% of which were unique. Similar to the targeted studies of the oral cavity, some showed little evolution over time. [159]

CRISPR evolution was studied in chemostats using S. thermophilus to directly examine spacer acquisition rates. In one week, S. thermophilus strains acquired up to three spacers when challenged with a single phage. [160] During the same interval the phage developed single nucleotide polymorphisms that became fixed in the population, suggesting that targeting had prevented phage replication absent these mutations. [160]

Another S. thermophilus experiment showed that phages can infect and replicate in hosts that have only one targeting spacer. Yet another showed that sensitive hosts can exist in environments with high phage titres. [161] The chemostat and observational studies suggest many nuances to CRISPR and phage (co)evolution.

CRISPRs are widely distributed among bacteria and archaea [87] and show some sequence similarities. [133] Their most notable characteristic is their repeating spacers and direct repeats. This characteristic makes CRISPRs easily identifiable in long sequences of DNA, since the number of repeats decreases the likelihood of a false positive match. [162]

Analysis of CRISPRs in metagenomic data is more challenging, as CRISPR loci do not typically assemble, due to their repetitive nature or through strain variation, which confuses assembly algorithms. Where many reference genomes are available, polymerase chain reaction (PCR) can be used to amplify CRISPR arrays and analyse spacer content. [118] [127] [163] [164] [165] [166] However, this approach yields information only for specifically targeted CRISPRs and for organisms with sufficient representation in public databases to design reliable polymerase chain reaction (PCR) primers. Degenerate repeat-specific primers can be used to amplify CRISPR spacers directly from environmental samples amplicons containing two or three spacers can be then computationally assembled to reconstruct long CRISPR arrays. [166]

The alternative is to extract and reconstruct CRISPR arrays from shotgun metagenomic data. This is computationally more difficult, particularly with second generation sequencing technologies (e.g. 454, Illumina), as the short read lengths prevent more than two or three repeat units appearing in a single read. CRISPR identification in raw reads has been achieved using purely de novo identification [167] or by using direct repeat sequences in partially assembled CRISPR arrays from contigs (overlapping DNA segments that together represent a consensus region of DNA) [159] and direct repeat sequences from published genomes [168] as a hook for identifying direct repeats in individual reads.

Another way for bacteria to defend against phage infection is by having chromosomal islands. A subtype of chromosomal islands called phage-inducible chromosomal island (PICI) is excised from a bacterial chromosome upon phage infection and can inhibit phage replication. [169] PICIs are induced, excised, replicated and finally packaged into small capsids by certain staphylococcal temperate phages. PICIs use several mechanisms to block phage reproduction. In first mechanism PICI-encoded Ppi differentially blocks phage maturation by binding or interacting specifically with phage TerS, hence blocks phage TerS/TerL complex formation responsible for phage DNA packaging. In second mechanism PICI CpmAB redirect the phage capsid morphogenetic protein to make 95% of SaPI-sized capsid and phage DNA can package only 1/3rd of their genome in these small capsid and hence become nonviable phage. [170] The third mechanism involves two proteins, PtiA and PtiB, that target the LtrC, which is responsible for the production of virion and lysis proteins. This interference mechanism is modulated by a modulatory protein, PtiM, binds to one of the interference-mediating proteins, PtiA, and hence achieving the required level of interference. [171]

One study showed that lytic ICP1 phage, which specifically targets Vibrio cholerae serogroup O1, has acquired a CRISPR/Cas system that targets a V. cholera PICI-like element. The system has 2 CRISPR loci and 9 Cas genes. It seems to be homologous to the I-F system found in Yersinia pestis. Moreover, like the bacterial CRISPR/Cas system, ICP1 CRISPR/Cas can acquire new sequences, which allows phage and host to co-evolve. [172]

Certain archaeal viruses were shown to carry mini-CRISPR arrays containing one or two spacers. It has been shown that spacers within the virus-borne CRISPR arrays target other viruses and plasmids, suggesting that mini-CRISPR arrays represent a mechanism of heterotypic superinfection exclusion and participate in interviral conflicts. [166]

CRISPR gene editing Edit

CRISPR technology has been applied in the food and farming industries to engineer probiotic cultures and to immunize industrial cultures (for yogurt, for instance) against infections. It is also being used in crops to enhance yield, drought tolerance and nutritional value. [173]

By the end of 2014 some 1000 research papers had been published that mentioned CRISPR. [174] [175] The technology had been used to functionally inactivate genes in human cell lines and cells, to study Candida albicans, to modify yeasts used to make biofuels and to genetically modify crop strains. [175] Hsu and his colleagues state that the ability to manipulate the genetic sequences allows for reverse engineering that can positively affect biofuel production [176] CRISPR can also be used to change mosquitos so they cannot transmit diseases such as malaria. [177] CRISPR-based approaches utilizing Cas12a have recently been utilized in the successful modification of a broad number of plant species. [178]

In July 2019, CRISPR was used to experimentally treat a patient with a genetic disorder. The patient was a 34-year-old woman with sickle cell disease. [179]

In February 2020, progress was made on HIV treatments with 60-80% of the DNA removed in mice and some being completely free from the virus after edits involving both LASER ART, a new anti-retroviral therapy, and CRISPR. [180]

In March 2020, CRISPR-modified virus was injected into a patient's eye in an attempt to treat Leber congenital amaurosis. [181]

In the future, CRISPR gene editing could potentially be used to create new species or revive extinct species from closely related ones. [182]

CRISPR-based re-evaluations of claims for gene-disease relationships have led to the discovery of potentially important anomalies. [183]

CRISPR as diagnostic tool Edit

CRISPR associated nucleases have shown to be useful as a tool for molecular testing due to their ability to specifically target nucleic acid sequences in a high background of non-target sequences. In 2016, the Cas9 nuclease was used to deplete unwanted nucleotide sequences in next-generation sequencing libraries while requiring only 250 picograms of initial RNA input. [184] Beginning in 2017, CRISPR associated nucleases were also used for direct diagnostic testing of nucleic acids, down to single molecule sensitivity. [185] [186]

By coupling CRISPR-based diagnostics to additional enzymatic processes, the detection of molecules beyond nucleic acids is possible. One example of a coupled technology is SHERLOCK-based Profiling of IN vitro Transcription (SPRINT). SPRINT can be used to detect a variety of substances, such as metabolites in patient samples or contaminants in environmental samples, with high throughput or with portable point-of-care devices. [187] CRISPR/Cas platforms are also being explored for detection [188] [189] [190] [191] [192] and inactivation of SARS-CoV-2, the virus that causes COVID-19. [193]


Conclusions

The CRISPR-Cas systems are extremely variable in their gene composition, and most of the cas genes evolve fast compared to other genes in prokaryotes. Accordingly, the comparative analyses of the Cas protein sequences and structures present a history of progressive detection of increasingly subtle relationship leading to unification of protein families previously thought to be unrelated ([2, 5, 19, 20]. and see Figure 4). The observations described here take this unification a step further. In particular, we substantially expanded the class of RAMPs and showed that at a high level the Cas proteins can be classified into no more than a dozen major groups of families including the Cas1-Cas10 proteins, another group of small subunits (Cas11?) and additionally a few regulatory protein families such as csm6. The majority of the families that have been left with historical "legacy" names in the recently published CRISPR-Cas classification scheme [20] now can be assigned to well-defined, "numbered" groups of cas genes (see Additional File 7). The results of this analysis emphasize that the CRISPR-Cas systems are built around RRM domains that reach extreme diversification in the RAMPs. This diversity along with recombination between different CRISPR-Cas loci makes more detailed classification and functional prediction for the CRISPR-Cas systems in the rapidly growing collection of archaeal and bacterial genomes a difficult challenge.

The unification of numerous Cas proteins into the three major groups of RAMPs and the more tentative demonstration of the probable origin of large subunits of diverse CRISPR-Cas systems from CRISPR polymerases together suggest a simple scenario for the origin and evolution of the CRISPR-Cas machinery in thermophilic archaea. Under this scenario, the CRISPR-Cas systems started from a large protein that combined the polymerase and HD hydrolase domain and might have functioned as a stand-alone antivirus defense system. The next step of evolution might have involved duplication of the RRM portion of the polymerase followed by inactivation that produced the ancestral, Cas7-like RAMP or a recruitment of a distinct RRM-domain protein that became the ancestral RAMP. Regardless of the origin of the ancestral RAMP genes, it has undergone a series of additional duplications and rapid diversification that yielded the stand-alone Cascade complex. The formation of the ancestral CRISPR-Cas system was then completed through the unification of Cascade with Cas1 and Cas2. The central theme of this scenario is the origin of the components of the CRISPR-Cas system from different classes of mobile elements. Other prokaryotic defense systems such as restriction-modification [56, 57] and toxin-antitoxin systems [58, 59] also comprise of such elements, indicating a major trend in the relationships between prokaryotes, viruses that infect them, other classes of selfish element and defense mechanisms.


CRISPR-Cas System Mechanism

The CRISPR-Cas system functions through three different steps: adaptation (or spacer acquisition), crRNA maturation, and interference (Figs. 2 and 3).

Adaptation

In the adaptation stage, Cas1 and Cas2 are the two main protein components which play roles in three main types (I, II, and III) of CRISPR-Cas systems. Both of these proteins are dimers that can form a complex together in order to undergo acquisition of foreign DNA [30]. Cas1 has both nuclease and integrase activity and can cut the viral genome and integrate a specific piece of the viral genetic element into the spacer DNA, whereas Cas2 is an endoribonuclease that mainly cuts RNAs [30, 75]. Since the subtype I-E of the CRISPR-Cas system of E. coli has been well-studied and characterized, it has become the best model for analysis of the adaptation mechanism of type I systems. In CRISPR-Cas type I and II systems, a short sequence named the protospacer-adjacent motif (PAM) exists in the foreign genome that leads Cas1 and Cas2 to recognize the protospacer flanked region. In subtype I-E of E. coli, in the presence of PAM, Cas1 and Cas2 recognize the adjacent sequence to the PAM. Cas1 and Cas2 dimers cut the PAM-adjacent sequence. After adjusting the size of the protospacer, the presence of the integrated host factor (IHF) protein is required for integrating the spacer into the host genome. By bending the host DNA near the insertion site, IHF guides Cas1 and Cas2 to the correct position of the CRISPR array for integrating the spacer [30, 75]. However, in the CRISPR-Cas system of Streptococcus pyogenes (subtype II-A), other Cas proteins including Cas9, Csn2 (whose function is related to Cas1 and Cas2 in DNA acquisition process [85], trans-activating CRISPR RNA (tracrRNA) and the leader-anchoring site (LAS) element in place of IHF are required for correct integration of the spacer sequence by Cas1 and Cas2 [75].

CrRNA Maturation

In the crRNA maturation step, transcription begins in the CRISPR array. The RNA transcribed by this process is termed pre-crRNA and contains complementary sequences of the repeats at the 3′ end and the spacers at the 5′ end. The maturation process of pre-crRNA occurs differently in various types of CRISPR-Cas systems [75]. In type I CRISPR-Cas systems, palindromic repeat sequences of pre-crRNA located at the 3′ end of the transcript, transform these parts of pre-crRNA into a hairpin-like structure. Following pre-crRNA transcription, Cas6 endoribonuclease attaches to the hairpin-like structure and cuts the 5′ end of the spacer sequence adjacent to that hairpin-like sequence. At the end of this process, there are several mature crRNAs that enable a Cas6 protein to remain bound to each one. An exception to this is seen with processes involving types I-A and I-B where the repeat sequences are not palindromic and Cas6 releases the crRNA [30]. This product is a ribonucleoprotein that identifies a specific phage genome [29].

In type II CRISPR-Cas systems, tracrRNA binds to the repeat sequence on the crRNA and transforms the 3′end of crRNA into a double-strand RNA. This double-strand RNA is called single guide RNA (sgRNA). In this type, RNase III and Cas9 catalyze the cleavage of pre-crRNA that yield to maturation crRNAs [12].

In type III systems, a dimer of Cas6 cleaves the 3′end of the repeat sequences adjacent to the spacers within the pre-crRNA. Once the cleavage process is complete the mature crRNA is then released.

crRNA maturation in type IV CRISPR systems is unknown at present. Cas12 and Cas13 carry out the cleavage of pre-crRNA and maturation of crRNA in types V and VI, respectively [75].

Interference

During the final step of interference, type-specific Cas proteins together with crRNA form a complex that recognizes and cuts the invader’s genome. In types I and II, the PAM plays an important role to increase the specificity of recognizing the invader’s genetic elements because not only can the crRNA identify the phage’s DNA sequence but the Cas enzyme can distinguish the PAM sequence [80].

In type I systems, interference occurs through a complex involving crRNA and Cas6, which acts as a scaffold for attaching other Cas proteins (including Cas5, Cas7, Cas8, and Cas11) to form CRISPR-associated complex for antiviral defense (Cascade). This in turn recognizes the invader’s genome. Moreover, recognition of the PAM sequence by Cas8e as the large subunit of Cascade increases the specificity of target recognition. After binding between crRNA of complex and target DNA the complex recruits Cas3 which functions as the nuclease that cleaves the non-target DNA strand to make an intermediated product. The ultimate cleavage of target DNA seems to occur by another nuclease provided either by the host cell or by utilizing the cascade-independent activity of Cas3 [7, 30].

In type II interference, a complex consisting of sgRNA and Cas9 recognizes the target DNA and cleaves it. This particular types has been utilized extensively for the purpose of gene editing in addition to diagnostics [16].

Type III interference is similar to that of type I systems where a complex of Cas proteins (involving Cas5, Cas7, Cas10, and Cas11) named Csm (subtype III-A) and Cmr (subtype III-B) accompanied by crRNA, recognizes the RNA that is transcribed from target DNA. After binding to this single-strand RNA, cleavage is carried out by subunits of Cas7. Alternatively, Cas10 can catalyze dsDNA cleavage and by transforming ATP into cyclic AMP as a second messenger, can activate the Csm6 protein which is an RNase. Csm6 then cleaves remaining RNAs in a nonspecific manner [30].

Type IV interference has not undergone characterization [30]. Interference in type V systems is divided into three subtypes: A, B, and C, in which the effector proteins in interference processes are Cas12a, Cas12b, and Cas12c, respectively. Similar to Cas9, Cas12b and Cas12c need the tracrRNA for their interfering activity while Cas12a does not. Cas12 proteins have a collateral nuclease activity which has been used in diagnostic applications such as the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) method. During the interference step, recognizing the target double-strand DNA (dsDNA) and PAM sequence is accomplished by crRNA and Cas12, respectively. After binding this complex to the target, Cas12 cuts the dsDNA and by its collateral nuclease activity also cuts surrounding single-strand DNAs nonspecifically. Both of these nuclease activities are catalyzed by the RuvC domain of Cas12a protein [46].

In type VI system interference, Cas13, which has higher eukaryotes and prokaryotes nucleotide (HEPN)-binding domains [30], which has collateral nuclease activity like Cas12 acts as the effector protein. This system is utilized in specific high sensitivity enzymatic reporter unlocking (SHERLOCK) technology. Cas13 does not require tracrRNA and PAM. The target of the Cas13-crRNA complex is single-strand RNAs (ssRNAs). In addition to crRNA guidance, the Cas13-crRNA complex requires a protospacer flanking site (PFS) for binding to the complementary ssRNA. PFS is an analogue of the PAM sequences on the RNA targets and is required for Cas13a activity in Leptotrichia shahii [30]. After binding, the complex cleaves the target and non-target ssRNAs [46, 75].


Strategies to Increase Specificity

Controlling Cas9/sgRNA abundance and duration

Typically Cas9 and sgRNA are expressed in cells by transient transfection of expressing plasmids. Titrating down the amount of plasmid DNA used in transfection increases specificity, although there is a trade-off for decreased efficiency at the on-target site. This is particularly an issue when the promoter is very strong, i.e. successfully transfected cells express a large amount of Cas9 and sgRNA leading to off-target effects. More recently, sgRNA has been expressed by RNA Pol II transcription and processed from introns, microRNAs, ribozymes, and RNA-triplex-helix structures, providing more flexible control of the sgRNA abundance [64,65].

Alternative delivery methods have also been developed to increase specificity. Compared to plasmid transfection based delivery, direct delivery of recombinant Cas9 protein and in vitro transcribed sgRNA, either individually or as purified complex, reduces off-targets in cells [66,67]. This is likely due to the rapid degradation of the protein and RNA in cells, which would lower the effective concentration of the Cas9-sgRNA effector complex and its duration in cells.

Paired nickase

The Cas9 “nickase” generated by mutating only one nuclease domain can only cleave one strand of the target DNA, which creates a nick thought to be repaired efficiently in cells. When the nickase is targeted to two neighboring regions on opposite strands, the offset double nicking leads to a double stranded break with tails that are degraded and subsequently indels in the target region. The requirement of dual Cas9 targeting to a nearby region dramatically increases the specificity, since it is generally unlikely that two guide RNAs will also have nearby off-targets. The limitation of this strategy is that nicks induced by Cas9 could still lead to mutations in off target sites via unknown mechanisms [13,29,60,63].

DCas9-FokI dimerization

FokI nuclease only cuts DNA when dimerized [68]. Fusion of dCas9 to FokI monomers creates an RNA-guided nuclease that only cuts the DNA when two guide RNAs bind nearby regions with defined spacing and orientation, thus substantially reducing off-target cleavage [69,70]. It has been reported that RNA-guided FokI nuclease is at least four fold more specific than paired Cas9 nickase [69], likely due to FokI nuclease only functioning when dimerized whereas Cas9 nickase can cleave as a monomer [70]. Similar to paired nickases, the requirement of two nearby PAM sites with defined spacing and orientation reduces the frequency of target sites in the genome.


Next Big Thing in Genome Modification: the CRISPR/Cas9 System

Ever since the discovery of the double-helix structure of DNA, scientists have sought ways to edit the genome. Altering gene expression partially and transiently via small interfering RNA has come a long way, and the progress has been spectacular. However, achieving complete and sustained modification of gene expression in a cell remains a tedious procedure that is often costly and time-consuming. For molecular biologists working with cell lines, quick and efficient knock out of one or more genes would provide a powerful tool for their studies. The CRISPR technology arrived two years ago to potentially fulfill that need.

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeat and is based on bacterial and archaeal adaptive immune systems. I recently used CRISPR technology in one of my experiments. My goal was to achieve efficient gene silencing of my gene of interest. Several attempts using transient transfection of various siRNAs had failed initially. While I was trying to understand what was going wrong and design new strategies, I went to a conference about RNA Biology. After talking with different fellow investigators, I received a lot of suggestions about using CRISPR technology.

The recently developed CRISPR/Cas technology enables precision genome modification.

A New Way to Get A Knockout

CRISPR elements were originally observed in E.coli and later in other bacteria and archaea as repetitive DNA elements where viral or other foreign DNA integrated. Once a foreign DNA is identified by the host cell, it is cleaved and produces short sequences (

20 nucleotides) that integrate into CRISPRs. Those new sequences are then transcribed into RNAs, which complex with Cas nucleases and scan the genome of viruses or other foreign DNA to guide the nucleases on the cleavage site and protect the host cell from the foreign genome. This process provides a quick response and destruction of viral DNA, acting as a molecular vaccine.

CRISPR takes advantage of its simple principle to target specific genes in the genome of eukaryotic cells for efficient gene knockout. A Cas9/single guide (sg)RNA sequence can be easily delivered into cells. Since Cas genes do not exist in eukaryotic genomes, a Cas9 protein has to be ectopically expressed (expression of a gene in an abnormal place in an organism). Once a double-strand break is created by the nuclease, the Non-Homologous End Joining (NHEJ) machinery is recruited, causes insertions or deletions, and eventually changes the open reading frame of the gene. Taking it a step further, homology-directed repair can be used to insert any sequence that might be requested into the site of the break, thus allowing for inherent modifications in a simple and fast way. For further information, the CRISPR/Cas9 technology and its applications were recently reviewed.

Possibilities For Multiple Fields

The CRISPR technology is the next best thing in several fields. Whole-genome screening of cells by using lentiviral-based CRISPR libraries was recently published. Moreover, genome engineering in germ lines can lead to easier and more efficient generation of animal models (mice, rats, flies, nematodes, zebrafish), and it can allow knocking-out of multiple genes using different sgRNA, each targeting a different gene. Corrections of genetic mutations related to disease (e.g., the CFTR gene) also represent promising clinical applications of CRISPR. In addition, viral and pathogen gene disruption can be achieved to provide immunization and to render plants and animals resistant to pests and disease.

After the conference, I went back to lab with excitement and ready to learn about the new technology to make my experiment work. Eventually, with careful experimental design, I achieved the gene silencing I was looking for. It is a great feeling to reach your goal and see a working result after a few failed attempts. I really appreciated the breakthrough discovery of CRISPR/Cas9, and I am happy I had the opportunity to interact with other scientists to keep myself updated on the latest technologies.

To explore how researchers at the NIH IRP use the CRISPR technology, visit the “Research in Action” story and video featuring Dr. Rafael Casellas and read Dr. Karen Usdin’s blog entry on “Why Studying Bacteria Matters.”


CRISPR-Cas Systems - Biology

Streptococcus is a diverse genus of Gram-positive bacteria commonly found in multiple locations of their human and animal hosts e.g. the oral cavity and the upper respiratory tract. Many streptococci are commensals thus play a role in colonization resistance of the host. Others, however, are pathogenic and are responsible for a range of invasive and noninvasive diseases with high morbidity and mortality worldwide. Streptococci undergo frequent and extensive horizontal gene transfer leading to the emergence of drug resistant strains. This, combined with the current lack of effective anti-streptococcal vaccines, has led to a huge amount of research into the genomics to systems biology of these important bacteria.

This timely review, edited by Yuqing Li and Xuedong Zhou, provides a comprehensive overview of the current knowledge of the most important hot-topics in streptococcal research. Topics covered include: CRISPR-Cas systems genetic regulation by small RNAs signal transduction by cyclic dinucleotide second messengers the VicRK and ComDE two-component systems mobile genetic elements regulation of cell division application of omics and bioinformatics tools intrageneric and intergeneric interactions by oral streptococci the hypervirulent M1T1 clone of GAS Streptococcus suis adhesion and invasion mechanisms molecular evolution of pathogenic streptococci novel antimicrobials vaccine development.

This volume is essential reading for everyone working with streptococci from the PhD student to the experienced scientist, in academia, the pharmaceutical or biotechnology industries and for those working in clinical environments.

(EAN: 9781912530229 9781912530236 Subjects: [bacteriology] [medical microbiology] [microbiology] [molecular microbiology] )


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Systems Biology 2014 - Topical Conference at the 2014 AIChE Annual Meeting. AIChE, 2014. p. 3-12 (Systems Biology 2014 - Topical Conference at the 2014 AIChE Annual Meeting).

Research output : Chapter in Book/Report/Conference proceeding › Conference contribution

T1 - Homology-integrated CRISPR-cas (HI-CRISPR) system for one-step multigene disruption in saccharomyces cerevisiae

N2 - One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.

AB - One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.

KW - Multiple gene disruption

KW - Saccharomyces cerevisiae

M3 - Conference contribution

T3 - Systems Biology 2014 - Topical Conference at the 2014 AIChE Annual Meeting

BT - Systems Biology 2014 - Topical Conference at the 2014 AIChE Annual Meeting

T2 - Systems Biology 2014 - Topical Conference at the 2014 AIChE Annual Meeting