CRISPR

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Type I CRISPR surveillance complex (Cas, blue) with bound target DNA (orange)

CRISPR ( C lustered R egularly I nterspaced S hort P alindromic R epeats) are portions to repetitive DNA ( repeats ), which in the genome of many bacteria and archaea occur. They serve a mechanism, the CRISPR / Cas system, which provides resistance to the penetration of foreign genes by viruses or plasmids , and are therefore part of the immune system equivalent of many prokaryotes . This system forms the basis of the genetic engineering CRISPR / Cas method for generating genetically modified organisms .

Discovery and Properties

The existence of repetitive stretches of DNA, now known as CRISPR, was discovered in 1987 in the bacterial strain Escherichia coli K12 by Yoshizumi Ishino and colleagues. They identified a repeating sequence of 29 nucleotides that were interrupted by the variable regions of 32 nucleotides. In 1993, similar regions were discovered on the DNA of Mycobacterium tuberculosis and referred to as "Direct Variable Repeats" (DVR). In 1995, these sequences were also discovered in the marine bacteria Haloferax volcanii and Haloferax mediterranei by the Spanish microbiologist Francisco Mojica , who called them " Tandem Repeats ”(TREPs). The Mojica working group identified other bacteria and archaea with corresponding sequences and chose a new name for these identical repetitions as “Short Regularly Spaced Repeats” (SRSR). In the literature, other names were added that also referred to these sequences, such as "spacer interspersed and direct repeats" (SPIDRs) and "long clustered tandem repeats" (LCTRs). In 2002, Jansen and colleagues first used the term “Clustered Regularly Interspaced Short Palindromic Repeats”, or CRISPR for short. It became known that similar structures exist in the genome of many different prokaryotes, and a group of genes was discovered that were close to the CRISPR gene locus in all of the organisms examined and were therefore called cas genes (CRISPR-associated). Jansen and colleagues identified four different Cas core sequences (Cas1 to Cas4); by 2005, Haft and colleagues had identified a total of 41 corresponding genes and two further Cas core sequences (Cas5 and Cas6) and a total of eight subtypes of CRISPR / Cas- Systems.

Today it is known that the genome of around 45% of the bacteria sequenced so far and 83% of the archaea contain at least one CRISPR structure.

Pathogens of the species Francisella use the CRISPR-Cas system for immune evasion . In Neisseria meningitidis and Campylobacter jejuni , the system is a pathogenicity factor with a previously unknown mechanism.

structure

Simplified diagram of the CRISPR gene locus. The gray boxes represent the repeats and the colored lines represent the spacers. The arrangement can vary in different organisms.

The CRISPR locus consists essentially of two main components: the cas genes containing cas - Operon and the CRISPR array, which is composed of a leader composed (also called repeat-spacer-array) sequence, and a repeat spacer sequence.

Repeat spacer sequence

The individual sequences of the repeating basic motif ( repeats ) have a length that varies between 23 and 47 bp . The repeats alternate with spacers that are 21 to 72 bp in length. While the repeating sequence is retained within a CRISPR structure, the sequence of the CRISPR varies greatly in different microorganisms. The sequence of CRISPR repeats of the bacteria is usually palindromic (i.e. mirror-inverted complementary), which results in a stable secondary structure of the associated RNA , whereas most repeats of the archaea are not palindromic.

The sequences of the spacer sections vary widely, both within a CRISPR structure and in different prokaryotes . In 2005 it was discovered that the spacer sequences are identical to foreign DNA from bacteriophages and plasmids. This led to the hypothesis that the function of CRISPR is to defend the organism against foreign DNA.

cas operon

The cas operon is also part of the CRISPR gene locus . The cas operon contains cas genes and the coding proteins that are necessary for the adaptive immune response, e.g. B. helicases , nucleases , but also proteins with properties for RNA binding. cas -Gene can be divided into two modules: the effector and the adaptation module . An effector module is a group of cas genes that is used to identify genetic material . The adaptation module also contains cas genes and, with the help of effector proteins, contributes to the selection of protospacers that can be integrated into the bacterial genome.

leader sequence

In the vicinity of the repeat spacer sequence there is a so-called leader sequence (not to be confused with the leader sequence of the mRNA). The leader sequence is an adenine and thymine- rich sequence with a length of 100-500 bp. As with the repeats, leader sequences are approx. 80% identical within a genome, but show significant differences within different organisms. As a non-coding sequence, it can be divided into two areas: a core leader and an extended leader . The core leader is preserved in several organisms and with a length of 20–300 bp is usually shorter than the extended leader . In addition, the core leader has a promoter element to which regulatory proteins can bind in order to be able to control gene expression , more precisely the initiation of CRISPR transcription , and spacer acquisition.

The extended leader with a length of 50-500 bp longer than the core leader and also contains in the CRISPR-distant regions of conserved sequences, presumably through gene duplication have been executed. The functions of the extended leader are currently unknown. The extended leader probably has no important functions.

Immunity through CRISPR

The CRISPR gene locus confers adaptive immunity to bacteria against repeated phage infections.
Transcripts of the CRISPR gene locus and maturation of the pre-crRNA.

The immunity through CRISPR takes place in three steps, whereby the last two steps are different for the respective CRISPR / Cas system types.

1. Adaptation: In 2007, Barrangou et al. Showed that bacteria that are infected with phages can integrate parts of the foreign DNA as spacers in the CRISPR regions of their genome and thereby develop immunity against the phages . They also showed that spacer sequences that are artificially inserted into the CRISPR regions of bacteria make them resistant to the associated phages. If the spacer sequences are cut out again, the resistance is also eliminated. It was also shown that the cas genes play an essential role in phage defense: the inactivation of some cas genes (cas1) prevents phage defense despite the presence of spacers. The activity of other cas genes (cas7) is necessary for the integration of new spacers in the CRISPR sequence.

2. crRNA transcription and processing: The CRISPR gene locus is transcribed to pre-crRNA and then processed to mature crRNA.

3. Interference: The mature crRNA is associated with a Cas protein or a Cas protein complex and this leads to the formation of an interference complex. With the CRISPR / Cas system types I and II, interaction of the interference complex with the PAM sequence motif of the phage DNA leads to the degradation of the DNA with the help of Cas3 for type I and Cas9 for type II, whereas type III does not require PAM and RNA can be broken down as well as DNA.

In the course of coevolution , anti-CRISPR proteins were developed by bacteriophages to inhibit the immune system.

mechanism

Overview of the three phases of the CRISPR / Cas9 process (based on Doudna & Charpentier 2014)

Despite great advances in recent years, the mechanism by which the CRISPR / Cas system provides immunity to prokaryotes is not yet fully understood. It is assumed that in the immunization process the exogenous DNA is recognized by a Cas protein complex and integrated into the CRISPR areas as a new spacer. How these processes work in detail is not yet fully understood.

adaptation

CRISPR / Cas systems are able to modify the genome of bacteria and archaea by integrating foreign DNA sequences, so-called spacers, between the repeats of the CRISPR array. This process is known as adaptation or spacer acquisition. The adaptation can be divided into two phases:

  1. Capture of spacer sequences of foreign DNA (so-called protospacers),
  2. Spacer integration.

The mechanism of adaptation has, with a few exceptions, been studied in detail in the E. coli CRISPR / Cas system type I (also known as the CRISPR / Cas system type IE). The main actors of the adaptation are encoded by the genes cas1 and cas2 , which are conserved in different CRISPR / Cas system types.

The first phase of adaptation, the capture of spacer sequences from the foreign DNA, can take place in two modes with the CRISPR / Cas system type I: naive or primed . In naive adaptation , only the proteins Cas1 and Cas2 are needed to capture spacers without prejudice, whereas primed adaptation depends on existing spacers ( priming spacers ) and a pre-selection is made as to which spacers are integrated into the genome. In addition to the Cas1 and Cas2 proteins, a protein complex made up of Cas proteins (interference complex type I, Cascade ) and the Cas3 nuclease are also required for this. Other types of CRISPR / Cas systems encode additional proteins for adaptation.

The mechanism of primed adaptation begins with the binding of the crRNA-bound protein complex Cascade ( CRISPR-associated complex for antiviral defense ) to the Protospacer Adjacent Motif (PAM) of the invading DNA by means of a combination of facilitated 1D diffusion (sliding along the DNA) and 3D diffusion ( hopping ). After the DNA has been bent and untwisted by Cascade, an R-loop is formed through complementary base pairing of the crRNA and the Cascade-bound DNA strand . When the Protospacer is completely unwound by Cascade, the R-Loop is completely formed. The complete formation of the R-loop leads to a change in the conformation of Cascade and thus causes Cas3 to bind to Cascade. In addition, the complete formation of the R-loop triggers a bulge on the unbound strand and thus enables the cut through Cas3 at this bulge. The single-stranded fragments generated by Cas3 are then processed into single-stranded protospacers by the Cas1-Cas2 complex. After processing into a single-stranded protospacer, it is converted into a complete or partially double-stranded protospacer, so that integration into the CRISPR array is possible. Even after the last step of immunity through CRISPR, the interference, the capture of spacer sequences is possible. The fragments of the degraded DNA are converted into protospacers by the enzyme RecBCD or other nucleases and integrated into the CRISPR array with the help of the Cas1-Cas2 complex (naive adaptation).

The spacer integration does not occur randomly in the CRISPR array, but is polarized, i.e. H. that spacers are integrated at a specific point in the CRISPR array, more precisely in the vicinity of the leader sequence. This mechanism ensures that new spacers are always integrated in the vicinity of the leader sequence and the chronological integration of the spacers optimizes the adaptive immune response to the most recent viral infections. With the CRISPR / Cas system type I, the protein integration host factor (IHF) is required, which can bind to the leader sequence. This bends the leader sequence by about 120 ° and creates a binding site for the Cas1-Cas2 complex, so that the complex is located in the vicinity of the repeat that is located closest to the leader sequence. As a result, the leader- repeat boundary becomes the place of spacer integration. With the CRISPR / Cas system type II, the spacer integration is also polarized, but without the use of additional proteins. The α-helix of Cas1 of the Cas1-Cas2 complex type II binds to the small groove of the leader sequence, which is also known as the leader-anchoring sequence (LAS). Due to the flexibility of the LAS-interacting domain of Cas1, the spacer integration does not necessarily have to take place at the leader- repeat boundary, but can also take place at a spacer-repeat boundary. In the case of a mutated LAS, this can lead to ectopic spacer integration, with spacers being integrated in the middle of the CRISPR array.

In E. coli , the spacer integration takes place through two transesterifications , the first transesterification taking place through the nucleophilic attack of the hydroxyl group at the 3 'end of one strand of the protospacer at the leader- repeat border and thereby leading to the formation of a half-site - Integration intermediary leads. The first transesterification creates an inflection of the repeat that enables a second transesterification. The transition to the fully integrated spacer, the full-site product, takes place through a second transesterification, with the nucleophilic attack of the hydroxyl group at the 3 'end of the opposite strand of the protospacer in the vicinity of the repeat-spacer boundary. The second transesterification is regulated by a so-called ruler mechanism. In E. coli , the repeat contains two inverse repeats (IR) that code for structural motifs and serve as anchors for so-called “molecular rulers”. These molecular rulers ensure that the second nucleophilic attack only takes place in the vicinity of the repeat-spacer boundary and that the length of the repeat is maintained after spacer integration and repeat duplication. The DNA gaps created after the transesterifications are closed by various DNA repair mechanisms , including homology-directed repair (HDR), non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ). After the spacer integration had taken place, the repeat adjoining the leader sequence was duplicated with the same length.

crRNA transcription and processing

The biogenesis of a mature CRISPR-RNA (crRNA) can take place in three steps and, with the help of its partially unique spacer sequence, leads one or more Cas proteins to the penetrating nucleic acid , which is used for possible degradation of the genetic material after sequence-specific RNA recognition.

  1. Transcription of a long primary transcript, the precursor crRNA (pre-crRNA), by a promoter located within the leader sequence.
  2. Primary cleavage of the pre-crRNA at specific sites to generate crRNA with an entire spacer sequence with partial repeat sequences.
  3. In some cases, an additional secondary cleavage is required to generate an active mature crRNA.

In the CRISPR / Cas systems I and III, a specific endoribonuclease of the Cas6 family or, alternatively, Cas5d for type IC, is required which, alone or in complex with other Cas proteins, cleaves the pre-crRNA within the repeat regions. In type II, a tracrRNA transactivates the cleavage of the pre-crRNA within the repeat regions by endoribonuclease III ( RNase III ) in the presence of Cas9.

With the CRISPR / Cas system type I, the processing of the pre-crRNA is catalyzed by endoribonucleases of the metal-independent Cas6 family (or alternatively with type IC by Cas5d), which place the repeat sequence at conserved positions, typically 8  nt upstream ("upstream") , in the direction of the 5 'end) from the repeat-spacer boundary, splits. While only one maturation step is required for types IC, IE and IF, a second maturation step is required for types IA, IB and ID, the components and mechanism of which are currently unknown. The palindromic repeats of the pre-crRNA of the types IC, ID, IE and IF have hairpin structures that expose the cleavage sites for the catalytic domain of the respective endoribonuclease. After cleavage, the hairpin structures remain associated with the respective endoribonuclease and the subunits of Cascade bind to the sequence at the 5 'end and to the spacer, which are used to recognize genetic material.

In the CRISPR / Cas system type II, after transcription of the CRISPR array and of tracrRNA, base pairing of the anti-repeat of tracrRNA with the repeat of the pre-crRNA takes place and the formation of the tracrRNA: pre-crRNA duplex, which is stabilized by Cas9 . In addition, the duplex formation leads to the recruitment of RNase III and thus to the co-processing of the duplex. This is followed by the second maturation step , with trimming by an exonuclease and / or cleavage by an endoribonuclease. Type II-C represents an alternative synthesis route for a mature crRNA. The promoters are located within the repeats of the CRISPR array and a short pre-crRNA transcript can be formed so that cleavage by RNase III is no longer necessary . The mature duplex is complexed with Cas9 and forms a type II interference complex that can recognize and cleave double-stranded DNA (dsDNA).

In the CRISPR / Cas system type III, the cleavage of the pre-crRNA within the repeats takes place by Cas6 and thus generates crRNA intermediates that each have a partial sequence of the repeats of the pre-crRNA at their 5 'and 3' ends ( 1X intermediate). Thereafter, the 1X intermediate is complexed with the Csm complex in III-A and with the Cmr complex in III-B. The second maturation step then takes place by trimming at the 3 'end using nucleases, which have not yet been identified, to form the mature crRNA.

interference

The after processing of the pre-crRNA to mature crRNA, which contain the integrated viral spacer sequences, associate with a CRISPR ribonucleoprotein complex (crRNP) and form an interference complex (also known as CRISPR surveillance complex) with the after a further infection, the viral DNA or RNA can be degraded sequence-specifically. The interference mechanism in all CRISPR / Cas system types is characterized by certain key proteins: Cas 3 (type I), Cas 9 (type II) and Cas10 (type III) and differ mainly in the structure of the crRNP complex (crRNP assembly ) and in the mechanism of degradation of genetic material. All crRNP complexes in type I are called cascades, whereas in type II the protein Cas9 as a single protein is responsible for the cleavage of the nucleic acid. In type III, the crRNP complexes Csm (type III-A) and Cmr (type III-B) are responsible for the interference.

With the CRISPR / Cas system type I, the interference occurs in five steps:

Drawing of the cascade surveillance complex of the bacterial CRISPR / Cas system type I of E. coli , which consists of cascade ( blue ) and the crRNA ( red ); according to PDB  4TVX .
  1. Cascade assembly
  2. PAM detection and retention
  3. R-loop formation
  4. Cas3 recruitment
  5. DNA degradation

After processing the pre-crRNA, the mature crRNA of E. coli consists of a 5 ' handle (8 nt) with a hydroxyl group, a spacer sequence (32 nt) and a hairpin structure at the 3' end (21 nt) a 2'-3'-cyclic phosphate end, with Cas6e remaining associated with the hairpin structure after processing. After cleavage of the mature crRNA, the cascade assembly takes place, the first step being termini capping . Cas5 binds to the 5'- handle and thus initially creates a hook-like structure of the crRNA. In addition, six copies of the Cas7 protein bind to the spacer sequence, resulting in the so-called Cas7 backbone . What is special is that the structures of Cas5 and Cas7 have a so-called conserved “palm-thumb domain”, which contribute to the interweaving of the Cas7 backbone. The “thumb” (a β-hairpin structure) of either Cas5e or of each of the six Cas7 subunits (Cas7.1 – Cas7.6) kinks the crRNA on the 5′- handle at a certain position and at 6-nt intervals within the spacer sequence and ensures that the kinked nucleotides adopt a deformed configuration and are no longer suitable for base pairing with the target DNA. In contrast, the adjacent 5-nt sequences protrude at every bend and retain their discontinuous A-DNA -like shape, so that these sequences are suitable for base pairing with the target DNA. Two other proteins, Cse1 (large subunit) and the Cse2 dimer (small subunits), then bind to the Cas7 subunits by means of protein-protein interaction . Both proteins are involved in DNA binding, with the large subunit also contributing to target selection. This ensures that the interference complex searches the cell for potential target DNA at all times. After the final assembly, Cascade is often described as a seahorse- like structure.

Now, with the help of Cascade, the search for the target DNA takes place, with the L1 loop of Cse1 being responsible for PAM identification. In type IE, after PAM identification, the double-stranded viral DNA enters the gap between Cas7.5 and Cas7.6 and is then passed on to the large subunit (Cse1), which, however, mainly has non-specific interactions with the target DNA. The PAM recognition by the L1 loop of Cse1 causes a destabilization of the double-stranded DNA, so that the base pairing between the 7 nt long seed region of the PAM-bordering DNA protospacer sequence with the crRNA can take place. The subsequent formation of an R-loop with complete base pairing of the crRNA spacer with the viral protospacer takes place according to the same mechanism as when capturing spacer sequences. After complete R-loop formation, the conformation of the large and small subunits changes, so that interaction sites are created on the large subunit for the C -terminal domain (CTD) of Cas3. Recruiting Cas3 at the bifurcation opens the channel for the double-stranded DNA by dissociating the CTD. After the dsDNA has accumulated in the channel, the channel is closed by repositioning the CTD and the unbound strand of the double-stranded DNA is stored in the HD nuclease domain of Cas3, where the cut takes place. The cut is made approximately 11–15 nt downstream (“downstream”, towards the 3 'end) of the PAM with the help of two catalytic transition metal ions. The conformational change of Cas3 in the helica part triggered by the cut (consisting of the RecA-like domain ( RecA ) and the RecA-like domain 2 ( RecA2 )) causes ATP binding and hydrolysis , the energy released for unwinding the dsDNA in 3 ′ → 5 ′ direction is used. The unwinding takes place on a hairpin structure of RecA2. By moving the helica part, this triggers a shift of the HD domain to new substrates for further exonucleolytic degradation. The single-stranded DNA (ssDNA) formed after degradation are also exonucleolytically degraded by Cas3. Thus, the target DNA can be effectively removed from the cell and Cascade can be recycled for further PAM recognition.

With the CRISPR / Cas system type II, the interference occurs in four steps:

Drawing of the CRISPR surveillance complex of the bacterial CRISPR / Cas system type II, which consists of Cas9 ( light blue ) and the crRNA ( red ) and is bound to DNA ( yellow ); according to PDB  4UN3 .
  1. Formation of the active type II CRISPR surveillance complex
  2. PAM detection and retention
  3. R-loop formation
  4. DNA degradation

Three independent studies on the structure of Cas9 by S. pyogenes show that Cas9 consists of two lobes that together adopt a crescent moon conformation. The REC lobe (English recognition lobe ) consists of a long α-helix (bridge helix), a Rec2 domain and a Rec1 domain to recognize the tracrRNA: crRNA duplex. The NUC lobe (English nuclease lobe ) consists of two nuclease domains for DNA cleavage, which are called HNH (named after characteristic histidine and asparagine residues ) and RuvC (named after an E. coli protein that is attached to the DNA participates repair) are known, and an additional C -terminal topoisomerase - homology domain (CTD), which is necessary to facilitate the PAM recognition.

The activation of Cas9 by binding the duplex to Rec1 before the interference triggered a conformational change of HNH, which led to the change in position of the REC lobe and the formation of a central positively charged channel for the invading DNA. The active type II CRISPR surveillance complex formed after co-processing the duplex is now ready to search for a viral DNA with a PAM sequence. After the PAM binding, local melting of the DNA occurs. Here unpaired are nucleic bases , so-called molten bubbles (engl. Melted bubbles ) formed nt to R-loop formation at a PAM proximal 8-12 long seed contribute sequence of the DNA. Each nuclease domain then cleaved a DNA strand in the presence of Mg 2+ ions, the HNH domain cleaving the target DNA strand hybridized to the crRNA and the RuvC domain cleaving the unhybridized DNA strand. The resulting cut, which takes place about 3 nt up the strand from the PAM, leads to the formation of double strand breaks with blunt ends (English for "smooth end"). Thereafter, Cas9 remains firmly associated with the blunt ends of the viral DNA.

In type III CRISPR / Cas systems, the interference complex recognizes the resulting RNA transcript, which is complementary to the sequence of the crRNA spacer, and degrades both the transcript and the DNA from which the transcript arose. This process is known as transcription-dependent DNA interference. The interference complex has three enzymatic activities:

  1. crRNA-directed endoribonuclease activity against the target RNA by Csm3 (type III-A) or Cmr4 (type III-B)
  2. Target RNA-stimulated DNase activity through the HD domain of Cas10 (Csm1 in types III-A and III-D or Cmr2 in types III-B and III-C)
  3. Target-RNA-stimulated cOA (cyclic oligoadenylate) synthetase activity through the "palm domain" of Cas10 (Csm1 for types III-A and III-D or Cmr2 for types III-B and III-C)

In bacteria, the crRNA-controlled complexes Csm (type III-A) or Cmr (type III-B) are brought to the RNA transcript, which triggers the cleavage of the transcript by the subunits Csm3 or Cmr4 and at the same time the DNase activity of Csm1 or Cmr2 activated for coupled degradation of ssDNA in the transcription bubble. The “palm domain”, more precisely the cyclase domain, of Csm1 or Cmr2 can produce cOA from ATP when the RNA transcript is bound. cOA in turn binds and activates the ribonuclease Csm6 (type III-A) or Csx1 (type III-B, III-C and III-D) to increase their ribonuclease activity in order to degrade RNA transcripts and thus form an additional interference Mechanism.

Effects

Through the CRISPR / Cas mechanism, bacteria can acquire immunity to certain phages and pass on the acquired immunity, as they integrate a virus-specific spacer into their genome and thus pass it on during replication . For this reason, the provocative thesis was expressed that the CRISPR-Cas system was the first really Lamarckist inheritance mechanism.

Applications

There are several suggestions for using CRISPR biotechnologically :

  • Artificial immunization against phages by adding suitable spacers to industrially important bacteria, e.g. B. in the dairy or wine industry ,
  • Knockdown of endogenous genes by transformation with a plasmid containing a CRISPR region with crRNA that matches the gene to be shut down,
  • Multiplex Genome Editing allows the simultaneous mutation of different target sequences, which shortens the production time of transgenic animals such as mice from up to two years to a few weeks,
  • Differentiation of different bacterial strains by comparing the spacer regions ( spoligotyping ),
  • Gene therapy ,
  • Fluorescent marking .

literature

Individual evidence

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