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What is the role of tracrRNA in CRISPR-cas9?

What is the role of tracrRNA in CRISPR-cas9?



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From what I understand, in a CRISPR cas9 complex, gRNA is comprised of tracrRNA and crRNA. I've read that crRNA is the part which is matched to the DNA which is targeted, but what role does tracrRNA play in the process? That I'm not clear about. Also, unless this should be its own question; how is gRNA prepared with cas9 protein? Does the protein automatically bind to certain RNA strands?


Your two questions are related and you are correct in your supposition that the Cas9 protein associates to a specific RNA sequence. That of the tracrRNA processed with the crRNA into a gRNA. This is achieved without any other aid so could be considered to do so automatically. How specific the tracrRNA sequence needs to be is an interesting and often overlooked question.

When comparing tracrRNA from different species it is clear that there is not a lot of sequence conservation but they are predicted to be contain similar secondary structures.

Matching tracrRNA and Cas9 from closely related species does result in in vitro DNA cleavage but this does not work with more distantly related species. Nevertheless they are remarkably versatile and this has been exploited in a number of exciting genome engineering applications where additional structures are added without compromising the binding of the RNA to the Cas9.

The tracrRNA and the crRNA together form the gRNA which targets the Cas9 protein to the location of cleavage. In the endogenous bacterial systems the gRNA takes the form of a processed RNA duplex but in the genome editing implementations this takes the form of a single chimeric RNA with the two components already fused together.

When it comes to actual cleavage of DNA it is important that the Cas9 is in the correct conformation and changes in this are known to occur when binding to the target DNA. Recent work suggests the tracrRNA plays a role in maintaining the Cas9 protein in an active form allowing it to be able to target DNA.

It likely also contributes to the overall stability (and thus efficiency) of the CRISPR-Cas9 complex.

Ultimately the tracrRNA is an essential component of the CRISPR-Cas9 system via it's role in both guiding the Cas9 protein to its target and aiding it's function.

The following references might be of interest to you:

The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems

Guide RNA engineering for versatile Cas9 functionality

Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease


In short, tracrRNA keeps the CRISPR-Cas9 complex (of RNA and the Cas9 protein) catalytically active:

Image source: Figure 5 from Lim, Y. et al., 2016. Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease. Nature Communications, 7, p.13350. Available at: http://dx.doi.org/10.1038/ncomms13350.


This was shown by pre-incubating different sets of components together for 20 minutes, then adding the missing component(s) and observing the resulting amount of DNA cleavage activity. For example, in the "pink" experiment, only the Cas9 protein and tracrRNA are pre-incubated together and the crRNA is added just before the 5 minute cleavage time:

Image source: Figure 1 from Lim, Y. et al., 2016. Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease. Nature Communications, 7, p.13350. Available at: http://dx.doi.org/10.1038/ncomms13350.

The green bar represents the Cas9 protein pre-incubated for 20 minutes with both the tracrRNA and crRNA. The pink bar shows that pre-incubating with tracrRNA but without crRNA still results in the same level of activity (measured as fraction of cleaved product). However, when the tracrRNA is not included in the pre-incubation, as in the yellow and blue experiments, the activity is much reduced.


CRISPR-Cas9 Structures and Mechanisms

Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

Keywords: CRISPR Cas9 genome engineering mechanism off-target structure.


Gene-editing 'scissor' tool CRISPR-Cas9 may also be a genetic 'dimmer switch'

In a series of experiments with laboratory-cultured bacteria, Johns Hopkins scientists have found evidence that there is a second role for the widely used gene-cutting system CRISPR-Cas9—as a genetic dimmer switch for CRISPR-Cas9 genes. Its role of dialing down or dimming CRISPR-Cas9 activity may help scientists develop new ways to genetically engineer cells for research purposes.

First identified in the genome of gut bacteria in 1987, CRISPR-Cas9 is a naturally occurring but unusual group of genes with a potential for cutting DNA sequences in other types of cells that was realized 25 years later. Its value in genetic engineering—programmable gene alteration in living cells, including human cells—was rapidly appreciated, and its widespread use as a genome "editor" in thousands of laboratories worldwide was recognized in the awarding of the Nobel Prize in Chemistry last year to its American and French co-developers.

CRISPR stands for clustered, regularly interspaced short palindromic repeats. Cas9, which refers to CRISPR-associated protein 9, is the name of the enzyme that makes the DNA slice. Bacteria naturally use CRISPR-Cas9 to cut viral or other potentially harmful DNA and disable the threat, says Joshua Modell, assistant professor of molecular biology and genetics at the Johns Hopkins University School of Medicine. In this role, Modell says, "CRISPR is not only an immune system, it's an adaptive immune system—one that can remember threats it has previously encountered by holding onto a short piece of their DNA, which is akin to a mug shot." These mug shots are then copied into "guide RNAs" that tell Cas9 what to cut.

Scientists have long worked to unravel the precise steps of CRISPR-Cas9's mechanism and how its activity in bacteria is dialed up or down. Looking for genes that ignite or inhibit the CRISPR-Cas9 gene-cutting system for the common, strep-throat causing bacterium Streptococcus pyogenes, the Johns Hopkins scientists found a clue regarding how that aspect of the system works.

Specifically, the scientists found a gene in the CRISPR-Cas9 system that, when deactivated, led to a dramatic increase in the activity of the system in bacteria. The product of this gene appeared to re-program Cas9 to act as a brake, rather than as a "scissor," to dial down the CRISPR system.

"From an immunity perspective, bacteria need to ramp up CRISPR-Cas9 activity to identify and rid the cell of threats, but they also need to dial it down to avoid autoimmunity—when the immune system mistakenly attacks components of the bacteria themselves," says graduate student Rachael Workman, a bacteriologist working in Modell's laboratory.

To further nail down the particulars of the "brake," the team's next step was to better understand the product of the deactivated gene (tracrRNA). RNA is a genetic cousin to DNA and is vital to carrying out DNA "instructions" for making proteins. TracrRNAs belong to a unique family of RNAs that do not make proteins. Instead, they act as a kind of scaffold that allows the Cas9 enzyme to carry guide RNA containing the mug shot of the invader virus and cut the virus' matching DNA sequences.

TracrRNA comes in two sizes: long and short. Most of the modern gene-cutting CRISPR-Cas9 tools use the short form. However, the research team found that the deactivated gene product was the long form of tracrRNA, the function of which has been entirely unknown.

The long and short forms of tracrRNA are similar in structure and have in common the ability to bind to Cas9. The short form tracrRNA also binds to the guide RNA. However, the long form tracrRNA doesn't need to bind to the guide RNA because it already contains a segment that mimics the guide RNA. "Essentially, long form tracrRNAs have combined the function of the short form tracrRNA and guide RNA," says Modell.

In addition, the researchers found that while guide RNAs normally seek out viral DNA sequences, long form tracrRNAs target the CRISPR-Cas9 system itself. The long form tracrRNA tends to sit on DNA, rather than cut it. When this happens in a particular area of a gene, it prevents that gene from expressing—or becoming functional.

To confirm this, the researchers used genetic engineering to alter the length of a certain region in long form tracrRNA to make the tracrRNA appear more like a guide RNA. They found that with the altered long form tracrRNA, Cas9 once again behaved more like a scissor.

Other experiments showed that in lab-grown bacteria with a plentiful amount of long form tracrRNA, levels of all CRISPR-related genes were very low. When the long form tracrRNA was removed from bacteria, however, expression of CRISPR-Cas9 genes increased a hundredfold.

Bacterial cells lacking the long form tracrRNA were cultured in the laboratory for three days and compared with similarly cultured cells containing the long form tracrRNA. By the end of the experiment, bacteria without the long form tracrRNA had completely died off, suggesting that long form tracrRNA normally protects cells from the sickness and death that happen when CRISPR-Cas9 activity is very high.

"We started to get the idea that the long form was repressing but not eliminating its own CRISPR-related activity," says Workman.

To see if the long form tracrRNA could be re-programmed to repress other bacterial genes, the research team altered the long form tracrRNA's spacer region to let it sit on a gene that produces green fluorescence. Bacteria with this mutated version of long form tracrRNA glowed less green than bacteria containing the normal long form tracrRNA, suggesting that the long form tracrRNA can be genetically engineered to dial down other bacterial genes.

The researchers also found the genetic components of long form tracrRNA in about 40% of the Streptococcus group of bacteria. Further study of bacterial strains that don't have the long form tracrRNA, says Workman, will potentially reveal whether their CRISPR-Cas9 systems are intact, and other ways that bacteria may dial back the CRISPR-Cas9 system.

The dimmer capability that the experiments uncovered, says Modell, offers opportunities to design new or better CRISPR-Cas9 tools aimed at regulating gene activity for research purposes. "In a gene editing scenario, a researcher may want to cut a specific gene, in addition to using the long form tracrRNA to inhibit gene activity," he says.


Prediction and diversity of tracrRNAs from type II CRISPR-Cas systems

Type II CRISPR-Cas9 systems require a small RNA called the trans-activating CRISPR RNA (tracrRNA) in order to function. The prediction of these non-coding RNAs in prokaryotic genomes is challenging because they have dissimilar structures, having short stems (3-6 bp) and non-canonical base-pairs e.g. G-A. Much of the tracrRNA is involved in base-pairing interactions with the CRISPR RNA, or itself, or in RNA-protein interactions with Cas9. Here we develop a new bioinformatic tool to predict tracrRNAs. On an experimentally verified test set the algorithm achieved a high sensitivity and specificity, and a low false discovery rate (FDR) on genome analysis. Analysis of representative RefSeq genomes (5462) detected 275 tracrRNAs from 165 genera. These tracrRNAs could be grouped into 15 clusters which were used to build covariance models. These clusters included Streptococci and Staphylococci tracrRNAs from the CRISPR-Cas9 systems which are currently used for gene editing. Compensating base changes observed in the models were consistent with the experimental structures of single guide RNAs (sgRNAs). Other clusters, for which there are not yet structures available, were predicted to form novel tracrRNA folds. These clusters included a large and divergent tracrRNA set from Bacteroidetes. These computational models contribute to the understanding of CRISPR-Cas biology, and will assist in the design of further engineered CRISPR-Cas9 systems. The tracrRNA prediction software is available through a galaxy web server.

Keywords: CM model CRISPR-Cas TracrRNA small RNA.

Figures

A schematic diagram of a type II CRISPR-Cas9 system focussing on the roles…

Features of type II interference…

Features of type II interference complexes, based mainly on sgRNA-Cas9 complexes and mutagenesis…

11–13 base-pairs but may be 15–25 in the native complex [4,5]. (ii) The nexus- consisting of unpaired bases, stem-loop 1 (SL1), non-canonical pairs (e.g. A-G) and part of the linker between SL1 and SL2 [4]. This region may form more complex structures e.g. a triple helix in C. jejuni [15] (iii) SL2- A second stem loop that may also include non-canonical pairs (A-G) [8]. SL3 – not predicted in all tracrRNAs nor found in all gRNA structures [18]. In gRNA complexes there are backbone and base interactions with the Cas9 protein and the three way helical junction, SL1 and the proximal part of SL2 [32] and with the bulged bases of the repeat-antirepeat hybrid [32]. The loops of the SLs are near the surface of the Cas9 complex.

A flow diagram showing the…

A flow diagram showing the process of tracrRNA prediction. (Results and Discussion and…

(A) Comparisons between tail-clusters. Similarities…

(A) Comparisons between tail-clusters. Similarities between clusters are calculated as link scores using…

tracrRNA tail clusters distribution across…

tracrRNA tail clusters distribution across genera. The relationships between the genera shown are…

Conserved sequences and structures in…

Conserved sequences and structures in tracrRNA tails. Bases that match to the structure…


Gene Editing

2 CRISPR/Cas Systems as Tools for Genome Editing

CRISPR/Cas systems are categorized into three types (I, II, and III), which are distinguished by different accessory RNAs and proteins. Of the three types, type II CRISPR/Cas systems have been identified for use in genome engineering. This choice stems from the fact that types I and III are composed of a number of Cas proteins, whereas the Cas9 protein is the only protein constituent of type II CRISPR/Cas systems ( Makarova et al., 2011 ). Thus design and construction of novel CRISPR/Cas systems with researcher-defined targeting sequences is simplified.

Preliminary proof of concept in vitro experimentation established the potential of CRISPR/Cas systems for genome editing ( Jinek et al., 2012 Gasiunas et al., 2012 ). Gasiunas and coworkers demonstrated the ability of a recombinant CRISPR/Cas system to cleave double-stranded DNA in which recognition is effected via RNA, and DSBs result from two domains in the Cas9 protein, each of which cleave a single DNA strand. Jinek and coworkers (2012) found that the Cas9 protein together with crRNA and an accessory RNA, the trans-activating crRNA (tracrRNA), could cleave DNA sequences that were homologous to the crRNA. Furthermore, when they created a chimeric RNA containing both crRNA and tracrRNA sequences on a single RNA molecule, they found the engineered single-RNA system just as capable of targeting specific DNA sequences as the wild-type 2-RNA systems ( Figure 8.4 ).

Figure 8.4 . CRISPR. A crRNA, consisting of a sequence homologous to the targeted genomic region and accessory RNA sequence, complexes with a Cas protein to produce a construct capable of recognizing and cleaving a targetable genomic locus.

The first cellular uses of CRISPR/Cas for genome engineering were described early in 2013 ( Cong et al., 2013 Mali et al., 2013 ). In these reports, mouse ( Cong et al., 2013 ) and human ( Cong et al., 2013 Mali et al., 2013 ) cells were transfected with DNAs coding for CRISPR RNAs and Cas9 protein and achieved mutational inactivation of multiple endogenous loci. Both groups developed CRISPR systems in which crRNA and tracrRNA were expressed as a single chimeric RNA (labeled gRNA by Mali et al., 2013 ), thus simplifying the construct assembly process. Additionally, in a subset of experiments a mutant version of Cas9 was used in both reports, in which only one strand of the target DNA was cleaved (as opposed to the wild-type activity of Cas9 which catalyzes the formation of DSBs). This creation of nicks in target loci restricted the resolution of genomic targeting events to HR only and not NHEJ.

Following these early accounts of the utility of CRISPR/Cas systems for genome editing, several groups have extended this technology to a number of animal species including zebrafish ( Hwang et al., 2013a,b Jao et al., 2013) , mouse ( Li et al., 2013a Menke, 2013 Wang et al., 2013b Yang et al., 2013 ), and rat ( Li et al., 2013a,c ).


Contents

To survive in a variety of challenging, inhospitable habitats that are filled with bacteriophages, bacteria and archaea have evolved methods to evade and fend off predatory viruses. This includes the CRISPR system of adaptive immunity. In practice, CRISPR/Cas systems act as self-programmable restriction enzymes. CRISPR loci are composed of short, palindromic repeats that occur at regular intervals composed of alternate CRISPR repeats and variable CRISPR spacers between 24-48 nucleotides long. These CRISPR loci are usually accompanied by adjacent CRISPR-associated (cas) genes. In 2005, it was discovered by three separate groups that the spacer regions were homologous to foreign DNA elements, including plasmids and viruses. These reports provided the first biological evidence that CRISPRs might function as an immune system.

Cas9 has been used often as a genome-editing tool. Cas9 has been used in recent developments in preventing viruses from manipulating hosts’ DNA. Since the CRISPR-Cas9 was developed from bacterial genome systems, it can be used to target the genetic material in viruses. The use of the enzyme Cas9 can be a solution to many viral infections. Cas9 possesses the ability to target specific viruses by the targeting of specific strands of the viral genetic information. More specifically the Cas9 enzyme targets certain sections of the viral genome that prevents the virus from carrying out its normal function. [13] Cas9 has also been used to disrupt the detrimental strand of DNA and RNA that cause diseases and mutated strands of DNA. Cas9 has already showed promise in disrupting the effects of HIV-1. Cas9 has been shown to suppress the expression of the long terminal repeats in HIV-1. When introduced into the HIV-1 genome Cas9 has shown the ability to mutate strands of HIV-1. [14] [15] Cas9 has also been used in the treatment of hepatitis b through targeting of the ends of certain of long terminal repeats in the hepatitis b viral genome. [16] Cas9 has been used to repair the mutations causing cataracts in mice.

CRISPR-Cas systems are divided into three major types (type I, type II, and type III) and twelve subtypes, which are based on their genetic content and structural differences. However, the core defining features of all CRISPR-Cas systems are the cas genes and their proteins: cas1 and cas2 are universal across types and subtypes, while cas3, cas9, and cas10 are signature genes for type I, type II, and type III, respectively.

CRISPR-Cas defense stages Edit

Adaptation Edit

Adaptation involves recognition and integration of spacers between two adjacent repeats in the CRISPR locus. The “Protospacer” refers to the sequence on the viral genome that corresponds to the spacer. A short stretch of conserved nucleotides exists proximal to the protospacer, which is called the protospacer adjacent motif (PAM). The PAM is a recognition motif that is used to acquire the DNA fragment. [7] In type II, Cas9 recognizes the PAM during adaptation in order to ensure the acquisition of functional spacers. [5]

CRISPR processing/biogenesis Edit

CRISPR expression includes the transcription of a primary transcript called a CRISPR RNA (pre-crRNA), which is transcribed from the CRISPR locus by RNA polymerase. Specific endoribonucleases then cleave the pre-crRNAs into small CRISPR RNAs (crRNAs). [17]

Interference/immunity Edit

Interference involves the crRNAs within a multi-protein complex called CASCADE, which can recognize and specifically base-pair with regions of inserting complementary foreign DNA. The crRNA-foreign nucleic acid complex is then cleaved, however if there are mismatches between the spacer and the target DNA, or if there are mutations in the PAM, then cleavage will not be initiated. In the latter scenario, the foreign DNA is not targeted for attack by the cell, thus the replication of the virus proceeds and the host is not immune to viral infection. The interference stage can be mechanistically and temporally distinct from CRISPR acquisition and expression, yet for complete function as a defense system, all three phases must be functional. [18]

Stage 1: CRISPR spacer integration. Protospacers and protospacer-associated motifs (shown in red) are acquired at the “leader” end of a CRISPR array in the host DNA. The CRISPR array is composed of spacer sequences (shown in colored boxes) flanked by repeats (black diamonds). This process requires Cas1 and Cas2 (and Cas9 in type II [5] ), which are encoded in the cas locus, which are usually located near the CRISPR array.

Stage 2: CRISPR expression. Pre-crRNA is transcribed starting at the leader region by the host RNA polymerase and then cleaved by Cas proteins into smaller crRNAs containing a single spacer and a partial repeat (shown as hairpin structure with colored spacers).

Stage 3: CRISPR interference. crRNA with a spacer that has strong complementarity to the incoming foreign DNA begins a cleavage event (depicted with scissors), which requires Cas proteins. DNA cleavage interferes with viral replication and provides immunity to the host. The interference stage can be functionally and temporarily distinct from CRISPR acquisition and expression (depicted by white line dividing the cell).

Transcription deactivation using dCas9 Edit

dCas9, also referred to as endonuclease deficient Cas9 can be utilized to edit gene expression when applied to the transcription binding site of the desired section of a gene. The optimal function of dCas9 is attributed to its mode of action. Gene expression is inhibited when nucleotides are no longer added to the RNA chain and therefore terminating elongation of that chain, and as a result affects the transcription process. This process occurs when dCas9 is mass-produced so it is able to affect the most amount of genes at any given time via a sequence specific guide RNA molecule. Since dCas9 appears to down regulate gene expression, this action is amplified even more when it is used in conjunction with repressive chromatin modifier domains. [19] The dCas9 protein has other functions outside of the regulation of gene expression. A promoter can be added to the dCas9 protein which allows them to work with each other to become efficient at beginning or stopping transcription at different sequences along a strand of DNA. These two proteins are specific in where they act on a gene. This is prevalent in certain types of prokaryotes when a promoter and dCas9 align themselves together to impede the ability of elongation of polymer of nucleotides coming together to form a transcribed piece of DNA. Without the promoter, the dCas9 protein does not have the same effect by itself or with a gene body. [20]

When examining the effects of repression of transcription further, H3K27, an amino acid component of a histone, becomes methylated through the interaction of dCas9 and a peptide called FOG1. Essentially, this interaction causes gene repression on the C + N terminal section of the amino acid complex at the specific junction of the gene, and as a result, terminates transcription. [21]

dCas9 also proves to be efficient when it comes to altering certain proteins that can create diseases. When the dCas9 attaches to a form of RNA called guide-RNA, it prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism's genome. Essentially, when multiple repeat codons are produced, it elicits a response, or recruits an abundance of dCas9 to combat the overproduction of those codons and results in the shut-down of transcription. dCas9 works synergistically with gRNA and directly affects the DNA polymerase II from continuing transcription.

Further explanation of how the dCas9 protein works can be found in their utilization of plant genomes by the regulation of gene production in plants to either increase or decrease certain characteristics. The CRISPR-CAS9 system has the ability to either upregulate or downregulate genes. The dCas9 proteins are a component of the CRISPR-CAS9 system and these proteins can repress certain areas of a plant gene. This happens when dCAS9 binds to repressor domains, and in the case of the plants, deactivation of a regulatory gene such as AtCSTF64, does occur. [22]

Bacteria are another focus of the usage of dCas9 proteins as well. Since eukaryotes have a larger DNA makeup and genome the much smaller bacteria are easy to manipulate. As a result, eukaryotes use dCas9 to inhibit RNA polymerase from continuing the process of transcription of genetic material. [23]

Crystal structure Edit

Cas9 features a bi-lobed architecture with the guide RNA nestled between the alpha-helical lobe (blue) and the nuclease lobe (cyan, orange, and gray). These two lobes are connected through a single bridge helix. There are two nuclease domains located in the multi-domain nuclease lobe, the RuvC (gray) which cleaves the non-target DNA strand, and the HNH nuclease domain (cyan) that cleaves the target strand of DNA. The RuvC domain is encoded by sequentially disparate sites that interact in the tertiary structure to form the RuvC cleavage domain (See right figure).

A key feature of the target DNA is that it must contain a protospacer adjacent motif (PAM) consisting of the three-nucleotide sequence- NGG. This PAM is recognized by the PAM-interacting domain (PI domain, orange) located near the C-terminal end of Cas9. Cas9 undergoes distinct conformational changes between the apo, guide RNA bound, and guide RNA:DNA bound states.

Cas9 recognizes the stem-loop architecture inherent in the CRISPR locus, which mediates the maturation of crRNA-tracrRNA ribonucleoprotein complex. [25] Cas9 in complex with CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) further recognizes and degrades the target dsDNA. [26] In the co-crystal structure shown here, the crRNA-tracrRNA complex is replaced by a chimeric single-guide RNA (sgRNA, in red) which has been proved to have the same function as the natural RNA complex. [4] The sgRNA base paired with target ssDNA is anchored by Cas9 as a T-shaped architecture. This crystal structure of the DNA-bound Cas9 enzyme reveals distinct conformational changes in the alpha-helical lobe with respect to the nuclease lobe, as well as the location of the HNH domain. The protein consists of a recognition lobe (REC) and a nuclease lobe (NUC). All regions except the HNH form tight interactions with each other and sgRNA-ssDNA complex, while the HNH domain forms few contacts with the rest of the protein. In another conformation of Cas9 complex observed in the crystal, the HNH domain is not visible. These structures suggest the conformational flexibility of HNH domain.

To date, at least three crystal structures have been studied and published. One representing a conformation of Cas9 in the apo state, [24] and two representing Cas9 in the DNA bound state. [27] [1]

Interactions with sgRNA Edit

In sgRNA-Cas9 complex, based on the crystal structure, REC1, BH and PI domains have important contacts with backbone or bases in both repeat and spacer region. [1] [27] Several Cas9 mutants including REC1 or REC2 domains deletion and residues mutations in BH have been tested. REC1 and BH related mutants show lower or none activity compared with wild type, which indicate these two domains are crucial for the sgRNA recognition at repeat sequence and stabilization of the whole complex. Although the interactions between spacer sequence and Cas9 as well as PI domain and repeat region need further studies, the co-crystal demonstrates clear interface between Cas9 and sgRNA.

DNA cleavage Edit

Previous sequence analysis and biochemical studies have posited that Cas9 contains two nuclease domains: an McrA-like HNH nuclease domain and a RuvC-like nuclease domain. [28] These HNH and RuvC-like nuclease domains are responsible for cleavage of the complementary/target and non-complementary/non-target DNA strands, respectively. [4] Despite low sequence similarity, the sequence similar to RNase H has a RuvC fold (one member of RNase H family) and the HNH region folds as T4 Endo VII (one member of HNH endonuclease family). [ citation needed ]

Wild-type S. pyogenes Cas9 requires magnesium (Mg 2+ ) cofactors for the RNA-mediated DNA cleavage however, Cas9 has been shown to exhibit varying levels of activity in the presence of other divalent metal ions. [4] For instance, Cas9 in the presence of manganese (Mn 2+ ) has been shown to be capable of RNA-independent DNA cleavage. [29] The kinetics of DNA cleavage by Cas9 have been of great interest to the scientific community, as this data provides insight into the intricacies of the reaction. While the cleavage of DNA by RNA-bound Cas9 has been shown to be relatively rapid (k ≥ 700 s −1 ), the release of the cleavage products is very slow (t1/2 = ln(2)/k ≈ 43-91 h), essentially rendering Cas9 a single-turnover enzyme. [30] Additional studies regarding the kinetics of Cas9 have shown engineered Cas9 to be effective in reducing off-target effects by modifying the rate of the reaction. [31] [32]

Problems bacteria pose to Cas9 editing Edit

Most archaea and bacteria stubbornly refuse to allow a Cas9 to edit their genome. This is because they can attach foreign DNA, that does not affect them, into their genome. Another way that these cells defy Cas9 is by process of restriction modification (RM) system. When a bacteriophage enters a bacteria or archaea cell it is targeted by the RM system. The RM system then cuts the bacteriophages DNA into separate pieces by restriction enzymes and uses endonucleases to further destroy the strands of DNA. This poses a problem to Cas9 editing because the RM system also targets the foreign genes added by the Cas9 process. [33]

Interference of transcription by dCas9 Edit

Due to the unique ability of Cas9 to bind to essentially any complement sequence in any genome, researchers wanted to use this enzyme to repress transcription of various genomic loci. To accomplish this, the two crucial catalytic residues of the RuvC and HNH domain can be mutated to alanine abolishing all endonuclease activity of Cas9. The resulting protein coined ‘dead’ Cas9 or ‘dCas9’ for short, can still tightly bind to dsDNA. This catalytically inactive Cas9 variant has been used for both mechanistic studies into Cas9 DNA interrogative binding and as a general programmable DNA binding RNA-Protein complex.

The interaction of dCas9 with target dsDNA is so tight that high molarity urea protein denaturant can not fully dissociate the dCas9 RNA-protein complex from dsDNA target. [34] dCas9 has been targeted with engineered single guide RNAs to transcription initiation sites of any loci where dCas9 can compete with RNA polymerase at promoters to halt transcription. [35] Also, dCas9 can be targeted to the coding region of loci such that inhibition of RNA Polymerase occurs during the elongation phase of transcription. [35] In Eukaryotes, silencing of gene expression can be extended by targeting dCas9 to enhancer sequences, where dCas9 can block assembly of transcription factors leading to silencing of specific gene expression. [10] Moreover, the guide RNAs provided to dCas9 can be designed to include specific mismatches to its complementary cognate sequence that will quantitatively weaken the interaction of dCas9 for its programmed cognate sequence allowing a researcher to tune the extent of gene silencing applied to a gene of interest. [35] This technology is similar in principle to RNAi such that gene expression is being modulated at the RNA level. However, the dCas9 approach has gained much traction as there exist less off-target effects and in general larger and more reproducible silencing effects through the use of dCas9 compared to RNAi screens. [36] Furthermore, because the dCas9 approach to gene silencing can be quantitatively controlled, a researcher can now precisely control the extent to which a gene of interest is repressed allowing more questions about gene regulation and gene stoichiometry to be answered.

Beyond direct binding of dCas9 to transcriptionally sensitive positions of loci, dCas9 can be fused to a variety of modulatory protein domains to carry out a myriad of functions. Recently, dCas9 has been fused to chromatin remodeling proteins (HDACs/HATs) to reorganize the chromatin structure around various loci. [35] This is important in targeting various eukaryotic genes of interest as heterochromatin structures hinder Cas9 binding. Furthermore, because Cas9 can react to heterochromatin, it is theorized that this enzyme can be further applied to studying the chromatin structure of various loci. [35] Additionally, dCas9 has been employed in genome wide screens of gene repression. By employing large libraries of guide RNAs capable of targeting thousands of genes, genome wide genetic screens using dCas9 have been conducted. [37]

Another method for silencing transcription with Cas9 is to directly cleave mRNA products with the catalytically active Cas9 enzyme. [38] This approach is made possible by hybridizing ssDNA with a PAM complement sequence to ssRNA allowing for a dsDNA-RNA PAM site for Cas9 binding. This technology makes available the ability to isolate endogenous RNA transcripts in cells without the need to induce chemical modifications to RNA or RNA tagging methods.

Transcription activation by dCas9 fusion proteins Edit

In contrast to silencing genes, dCas9 can also be used to activate genes when fused to transcription activating factors. [35] These factors include subunits of bacterial RNA Polymerase II and traditional transcription factors in eukaryotes. Recently, genome-wide screens of transcription activation have also been accomplished using dCas9 fusions named ‘CRISPRa’ for activation. [37]


Gene-editing 'scissor' tool may also be a 'dimmer switch'

In a series of experiments with laboratory-cultured bacteria, Johns Hopkins scientists have found evidence that there is a second role for the widely used gene-cutting system CRISPR-Cas9 -- as a genetic dimmer switch for CRISPR-Cas9 genes. Its role of dialing down or dimming CRISPR-Cas9 activity may help scientists develop new ways to genetically engineer cells for research purposes.

A summary of the findings was published Jan. 8 in Cell.

First identified in the genome of gut bacteria in 1987, CRISPR-Cas9 is a naturally occurring but unusual group of genes with a potential for cutting DNA sequences in other types of cells that was realized 25 years later. Its value in genetic engineering -- programmable gene alteration in living cells, including human cells -- was rapidly appreciated, and its widespread use as a genome "editor" in thousands of laboratories worldwide was recognized in the awarding of the Nobel Prize in Chemistry last year to its American and French co-developers.

CRISPR stands for clustered, regularly interspaced short palindromic repeats. Cas9, which refers to CRISPR-associated protein 9, is the name of the enzyme that makes the DNA slice. Bacteria naturally use CRISPR-Cas9 to cut viral or other potentially harmful DNA and disable the threat, says Joshua Modell, Ph.D., assistant professor of molecular biology and genetics at the Johns Hopkins University School of Medicine. In this role, Modell says, "CRISPR is not only an immune system, it's an adaptive immune system -- one that can remember threats it has previously encountered by holding onto a short piece of their DNA, which is akin to a mug shot." These mug shots are then copied into "guide RNAs" that tell Cas9 what to cut.

Scientists have long worked to unravel the precise steps of CRISPR-Cas9's mechanism and how its activity in bacteria is dialed up or down. Looking for genes that ignite or inhibit the CRISPR-Cas9 gene-cutting system for the common, strep-throat causing bacterium Streptococcus pyogenes, the Johns Hopkins scientists found a clue regarding how that aspect of the system works.

Specifically, the scientists found a gene in the CRISPR-Cas9 system that, when deactivated, led to a dramatic increase in the activity of the system in bacteria. The product of this gene appeared to re-program Cas9 to act as a brake, rather than as a "scissor," to dial down the CRISPR system.

"From an immunity perspective, bacteria need to ramp up CRISPR-Cas9 activity to identify and rid the cell of threats, but they also need to dial it down to avoid autoimmunity -- when the immune system mistakenly attacks components of the bacteria themselves," says graduate student Rachael Workman, a bacteriologist working in Modell's laboratory.

To further nail down the particulars of the "brake," the team's next step was to better understand the product of the deactivated gene (tracrRNA). RNA is a genetic cousin to DNA and is vital to carrying out DNA "instructions" for making proteins. TracrRNAs belong to a unique family of RNAs that do not make proteins. Instead, they act as a kind of scaffold that allows the Cas9 enzyme to carry the guide RNA that contains the mug shot and cut matching DNA sequences in invading viruses.

TracrRNA comes in two sizes: long and short. Most of the modern gene-cutting CRISPR-Cas9 tools use the short form. However, the research team found that the deactivated gene product was the long form of tracrRNA, the function of which has been entirely unknown.

The long and short forms of tracrRNA are similar in structure and have in common the ability to bind to Cas9. The short form tracrRNA also binds to the guide RNA. However, the long form tracrRNA doesn't need to bind to the guide RNA, because it contains a segment that mimics the guide RNA. "Essentially, long form tracrRNAs have combined the function of the short form tracrRNA and guide RNA," says Modell.

In addition, the researchers found that while guide RNAs normally seek out viral DNA sequences, long form tracrRNAs target the CRISPR-Cas9 system itself. The long form tracrRNA tends to sit on DNA, rather than cut it. When this happens in a particular area of a gene, it prevents that gene from expressing, -- or becoming functional.

To confirm this, the researchers used genetic engineering to alter the length of a certain region in long form tracrRNA to make the tracrRNA appear more like a guide RNA. They found that with the altered long form tracrRNA, Cas9 once again behaved more like a scissor.

Other experiments showed that in lab-grown bacteria with a plentiful amount of long form tracrRNA, levels of all CRISPR-related genes were very low. When the long form tracrRNA was removed from bacteria, however, expression of CRISPR-Cas9 genes increased a hundredfold.

Bacterial cells lacking the long form tracrRNA were cultured in the laboratory for three days and compared with similarly cultured cells containing the long form tracrRNA. By the end of the experiment, bacteria without the long form tracrRNA had completely died off, suggesting that long form tracrRNA normally protects cells from the sickness and death that happen when CRISPR-Cas9 activity is very high.

"We started to get the idea that the long form was repressing but not eliminating its own CRISPR-related activity," says Workman.

To see if the long form tracrRNA could be re-programmed to repress other bacterial genes, the research team altered the long form tracrRNA's spacer region to let it sit on a gene that produces green fluorescence. Bacteria with this mutated version of long form tracrRNA glowed less green than bacteria containing the normal long form tracrRNA, suggesting that the long form tracrRNA can be genetically engineered to dial down other bacterial genes.

Another research team, from Emory University, found that in the parasitic bacteria Francisella novicida, Cas9 behaves as a dimmer switch for a gene outside the CRISPR-Cas9 region. The CRISPR-Cas9 system in the Johns Hopkins study is more widely used by scientists as a gene-cutting tool, and the Johns Hopkins team's findings provide evidence that the dimmer action controls the CRISPR-Cas9 system in addition to other genes.

The researchers also found the genetic components of long form tracrRNA in about 40% of the Streptococcus group of bacteria. Further study of bacterial strains that don't have the long form tracrRNA, says Workman, will potentially reveal whether their CRISPR-Cas9 systems are intact, and other ways that bacteria may dial back the CRISPR-Cas9 system.

The dimmer capability that the experiments uncovered, says Modell, offers opportunities to design new or better CRISPR-Cas9 tools aimed at regulating gene activity for research purposes. "In a gene editing scenario, a researcher may want to cut a specific gene, in addition to using the long form tracrRNA to inhibit gene activity," he says.

Funding for the Johns Hopkins research was provided by the Johns Hopkins University School of Medicine.

Other scientists who contributed to the research include Teja Pammi, Binh Nguyen, Leonardo Graeff, Erika Smith, Suzanne Sebald and Marie Stoltzfus from Johns Hopkins, and Chad Euler from Weill Cornell Medical College.


CRISPR technology shown to dial down gene activity in bacteria

Left – a schematic of the long form of the tracrRNA used by the CRISPR-Cas9 system in bacteria Right – the standard guide RNA used by many scientists as part of the gene-cutting CRISPR-Cas9 system. Credit: Joshua Modell, Rachael Workman and Johns Hopkins Medicine

In a series of experiments with laboratory-cultured bacteria, Johns Hopkins scientists have found evidence that there is a second role for the widely used gene-cutting system CRISPR-Cas9—as a genetic dimmer switch for CRISPR-Cas9 genes. Its role of dialing down or dimming CRISPR-Cas9 activity may help scientists develop new ways to genetically engineer cells for research purposes.

A summary of the findings was published Jan. 8 in Cell.

First identified in the genome of gut bacteria in 1987, CRISPR-Cas9 is a naturally occurring but unusual group of genes with a potential for cutting DNA sequences in other types of cells that was realized 25 years later. Its value in genetic engineering—programmable gene alteration in living cells, including human cells—was rapidly appreciated, and its widespread use as a genome "editor" in thousands of laboratories worldwide was recognized in the awarding of the Nobel Prize in Chemistry last year to its American and French co-developers.

CRISPR stands for clustered, regularly interspaced short palindromic repeats. Cas9, which refers to CRISPR-associated protein 9, is the name of the enzyme that makes the DNA slice. Bacteria naturally use CRISPR-Cas9 to cut viral or other potentially harmful DNA and disable the threat, says Joshua Modell, Ph.D., assistant professor of molecular biology and genetics at the Johns Hopkins University School of Medicine. In this role, Modell says, "CRISPR is not only an immune system, it's an adaptive immune system—one that can remember threats it has previously encountered by holding onto a short piece of their DNA, which is akin to a mug shot." These mug shots are then copied into "guide RNAs" that tell Cas9 what to cut.

Scientists have long worked to unravel the precise steps of CRISPR-Cas9's mechanism and how its activity in bacteria is dialed up or down. Looking for genes that ignite or inhibit the CRISPR-Cas9 gene-cutting system for the common, strep-throat causing bacterium Streptococcus pyogenes, the Johns Hopkins scientists found a clue regarding how that aspect of the system works.

Specifically, the scientists found a gene in the CRISPR-Cas9 system that, when deactivated, led to a dramatic increase in the activity of the system in bacteria. The product of this gene appeared to re-program Cas9 to act as a brake, rather than as a "scissor," to dial down the CRISPR system.

"From an immunity perspective, bacteria need to ramp up CRISPR-Cas9 activity to identify and rid the cell of threats, but they also need to dial it down to avoid autoimmunity—when the immune system mistakenly attacks components of the bacteria themselves," says graduate student Rachael Workman, a bacteriologist working in Modell's laboratory.

To further nail down the particulars of the "brake," the team's next step was to better understand the product of the deactivated gene (tracrRNA). RNA is a genetic cousin to DNA and is vital to carrying out DNA "instructions" for making proteins. TracrRNAs belong to a unique family of RNAs that do not make proteins. Instead, they act as a kind of scaffold that allows the Cas9 enzyme to carry the guide RNA that contains the mug shot and cut matching DNA sequences in invading viruses.

TracrRNA comes in two sizes: long and short. Most of the modern gene-cutting CRISPR-Cas9 tools use the short form. However, the research team found that the deactivated gene product was the long form of tracrRNA, the function of which has been entirely unknown.

The long and short forms of tracrRNA are similar in structure and have in common the ability to bind to Cas9. The short form tracrRNA also binds to the guide RNA. However, the long form tracrRNA doesn't need to bind to the guide RNA, because it contains a segment that mimics the guide RNA. "Essentially, long form tracrRNAs have combined the function of the short form tracrRNA and guide RNA," says Modell.

In addition, the researchers found that while guide RNAs normally seek out viral DNA sequences, long form tracrRNAs target the CRISPR-Cas9 system itself. The long form tracrRNA tends to sit on DNA, rather than cut it. When this happens in a particular area of a gene, it prevents that gene from expressing,—or becoming functional.

To confirm this, the researchers used genetic engineering to alter the length of a certain region in long form tracrRNA to make the tracrRNA appear more like a guide RNA. They found that with the altered long form tracrRNA, Cas9 once again behaved more like a scissor.

Other experiments showed that in lab-grown bacteria with a plentiful amount of long form tracrRNA, levels of all CRISPR-related genes were very low. When the long form tracrRNA was removed from bacteria, however, expression of CRISPR-Cas9 genes increased a hundredfold.

Bacterial cells lacking the long form tracrRNA were cultured in the laboratory for three days and compared with similarly cultured cells containing the long form tracrRNA. By the end of the experiment, bacteria without the long form tracrRNA had completely died off, suggesting that long form tracrRNA normally protects cells from the sickness and death that happen when CRISPR-Cas9 activity is very high.

"We started to get the idea that the long form was repressing but not eliminating its own CRISPR-related activity," says Workman.

To see if the long form tracrRNA could be re-programmed to repress other bacterial genes, the research team altered the long form tracrRNA's spacer region to let it sit on a gene that produces green fluorescence. Bacteria with this mutated version of long form tracrRNA glowed less green than bacteria containing the normal long form tracrRNA, suggesting that the long form tracrRNA can be genetically engineered to dial down other bacterial genes.

Another research team, from Emory University, found that in the parasitic bacteria Francisella novicida, Cas9 behaves as a dimmer switch for a gene outside the CRISPR-Cas9 region. The CRISPR-Cas9 system in the Johns Hopkins study is more widely used by scientists as a gene-cutting tool, and the Johns Hopkins team's findings provide evidence that the dimmer action controls the CRISPR-Cas9 system in addition to other genes.

The researchers also found the genetic components of long form tracrRNA in about 40% of the Streptococcus group of bacteria. Further study of bacterial strains that don't have the long form tracrRNA, says Workman, will potentially reveal whether their CRISPR-Cas9 systems are intact, and other ways that bacteria may dial back the CRISPR-Cas9 system.

The dimmer capability that the experiments uncovered, says Modell, offers opportunities to design new or better CRISPR-Cas9 tools aimed at regulating gene activity for research purposes. "In a gene editing scenario, a researcher may want to cut a specific gene, in addition to using the long form tracrRNA to inhibit gene activity," he says.


DNA and RNA interference mechanisms by CRISPR-Cas surveillance complexes

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) adaptive immune systems use small guide RNAs, the CRISPR RNAs (crRNAs), to mark foreign genetic material, e.g. viral nucleic acids, for degradation. Archaea and bacteria encode a large variety of Cas proteins that bind crRNA molecules and build active ribonucleoprotein surveillance complexes. The evolution of CRISPR-Cas systems has resulted in a diversification of cas genes and a classification of the systems into three types and additional subtypes characterized by distinct surveillance and interfering complexes. Recent crystallographic and biochemical advances have revealed detailed insights into the assembly and DNA/RNA targeting mechanisms of the various complexes. Here, we review our knowledge on the molecular mechanism involved in the DNA and RNA interference stages of type I (Cascade: CRISPR-associated complex for antiviral defense), type II (Cas9) and type III (Csm, Cmr) CRISPR-Cas systems. We further highlight recently reported structural and mechanistic themes shared among these systems.

Keywords: CRISPR Cas9 Cascade DNA interference guide crRNAs ribonucleoprotein complexes tracrRNA viruses.

Figures

CRISPR-Cas systems and conserved stages…

CRISPR-Cas systems and conserved stages of CRISPR-Cas activity. The general organization of a…

Assembly of the type I-E Cascade structure. The I-E Cascade complex has a…

Structures of S. pyogenes (Spy)…

Structures of S. pyogenes (Spy) type II-A Cas9. ( A ) Crystal structure…

Structure of the DNA nuclease…

Structure of the DNA nuclease Cas3. The type I-E Cas3 crystal structure of…

Mechanism of type I Cascade-mediated…

Mechanism of type I Cascade-mediated DNA interference. After the assembly of the crRNA-loaded…

Mechanism of type II Cas9-mediated…

Mechanism of type II Cas9-mediated DNA interference. In the absence of type II…

Comparison of type III crRNP-mediated…

Comparison of type III crRNP-mediated RNA interference. ( A) The type III-A Csm…


How CRISPR Works

Illustration by Kelsey King Writer Maywa Montenegro
@MaywaMontenegro Food systems researcher, UC Berkeley

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January 28, 2016 &mdash Back in 2011, Jennifer Doudna, a biochemist and molecular biologist at the University of California, Berkeley, and Emmanuelle Charpentier, now at the Max Planck Institute for Infection Biology in Germany, grew intrigued by the way bacteria use a molecular system known as CRISPR-Cas9 to respond to viral attacks. For years, bacteria were assumed to be primitive creatures with rudimentary immune systems. But CRISPR-Cas9 revealed a startlingly sophisticated memory-response scheme. The bacteria store DNA samples from invading viruses by tucking them into a DNA library called CRISPR that is part of the bacteria’s natural genome. If the same virus should attack again, the Cas9 enzyme is primed by the CRISPR library to cut (and thus disable) viral DNA with the same sequence.

In the native bacterial system (a), a structure that’s formed by crRNA and tracrRNA and includes a “guide” segment (gold) guides the Cas9 protein (light blue blob) to a spot in the viral DNA that corresponds to the guide segment. The cRNA is critical in targeting while tracRNA stabilizes the structure and activates Cas9 to cleave the DNA. To turn this natural system into a useful tool for genetic manipulation (b), researchers created an artificial single guide RNA molecule (sgRNA, in green) by fusing the crRNA and tracrRNA. Image courtesy of Elsevier

After months of trying to tease apart how the system works, Doudna’s team determined that two RNA molecules play central roles: CRISPR RNA (crRNA), which leads Cas9 to a particular location on the viral gene, and a trans-activating RNA (tracrRNA), which helps activate Cas9. Together, these two RNA molecules empower Cas9 to make its cuts.

Still, it was not clear that CRISPR would be all that exciting or useful outside of bacteria. Microbes have very different cell structures than animals and plants, and it was quite possible that the system would only work in bacteria. The real breakthrough occurred in 2012 when Doudna, Charpentier and then-postdoctoral fellow Martin Jinek realized it would be possible to combine the crRNA and the tracrRNA into a single, artificial guide RNA (sgRNA). By adding to the sgRNA a customized “guide segment” matching a particular DNA sequence in an organism of interest, they could aim Cas9 to cut any organism’s genome in any spot they wished.

Often likened to a word processor, CRISPR can be used to target whole gene “words” or a few nucleotide “letters” with precision and speed that far outpaces conventional genetic engineering. It’s a superb tool for deleting chunks of DNA and for facilitating precise substitutions when researchers want to swap a few key nucleotide sequences.

Less often emphasized is that CRISPR can also be used to add new genes or parts thereof. The key here is understanding what happens after Cas9 makes its cuts.

A Cas9-caused break in DNA can be repaired in four different ways, two of which open the door to inserting a new gene of choice. Image courtesy of Elsevier

The cell’s DNA repair machinery typically takes over in one of two different modes. In the first mode (called “non-homologous end joining,” or NHEJ), it usually glues the two pieces back together, but imperfectly, deactivating the gene (see “a” above). Such “gene knockouts” don’t involve any foreign DNA but can eliminate traits that affect food quality, confer susceptibility to diseases or divert energy away from valuable end products such as grain or fruit. Occasionally, say researchers, this pathway may leave a DNA cut with “sticky ends,” enabling foreign genes of interest to be directly spliced in (b) — a double-stranded DNA insertion somewhat akin to “old-fashioned” genetic engineering.

A second kind of repair (called “homology-directed repair” or “homologous recombination” — HR) is much less common but far more accurate. In HR, the cut ends aren’t just jammed back together the cell machinery copies a nearby piece of DNA to fix the damaged sequence. By providing a DNA snippet of their choice, scientists can induce the cell to fill in any desired sequence, from a small mutation (c) to a whole new gene (d). This HR pathway, says Fuguo Jiang, a postdoctoral fellow in Doudna’s lab, is not yet fully understood. But, as this illustration shows, it involves a meticulous process of one strand of donor DNA being stitched into the host gene, providing the template for cellular repair.

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