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The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1)1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.
Design short Cas RNA substrates (e.g. single repeat sequences, Fig. 2C) and utilize custom RNA oligonucleotide synthesis facilities.
Note: The inclusion of a deoxyribonucleotide at a specified position of an RNA oligonucleotide (Fig. 2C) can be used to pinpoint the site of RNA cleavage.
An example of RNA substrates for the analysis of Cas endonuclease activity is shown in Figure 3A. An aliquot of 5 μl of an analytical 100 μl in vitro transcription reaction were loaded. Please note that the efficiency of RNA production varies between different constructs. Some factors that were observed to influence the amount of obtained RNA are (i) the initial sequence following the +1G required for transcription initiation, (ii) the possibility of RNA structure formation during transcription and (iii) the choice of the restriction site for the generation of the run-off cleavage position.
The investigation of RNA endonuclease activity requires both highly purified recombinant Cas proteins (Fig. 3B) and proper negative controls. Ideally, this negative control sample differs as little as possible from the investigated Cas endonuclease reaction. This can be achieved by incubation of the RNA with reaction buffer and cell-lysate without Cas expression (and following the identical purification procedure). An ideal negative control is the addition of a deoxyribonucleotide at the proposed cleavage site. In Figure 3C, the cleavage of a 5' terminal labeled repeat sequence is shown for Clostridium thermocellum Cas6. Under identical conditions, this repeat is not a substrate anymore when a deoxyribonucleotide is introduced at position -9. This method also provides information about the cleavage site. Finally, a long internally labeled pre-crRNA is cleaved by Cas6 and two cleavage fragments are observed.
Figure 1. Schematic overview of CRISPR/Cas activity. The overview follows the insertion of a viral DNA sequence (protospacer) into the CRISPR cluster (adaptation), the transcription and processing of the CRISPR array into small crRNAs by a Cas6 endonuclease, the uptake of crRNAs into the Cascade complex and the interference of a repeated viral attack based on complementarity between crRNA and protospacer. Protospacer adjacent motifs (PAM) mark viral protospacer sequences.
Figure 2. Generation of RNA substrates for Cas proteins. The scheme shows the workflow for generating (A) long pre-crRNA substrates, (B) intermediate pre-crRNA substrates and (C) short Cas RNA substrates for pre-crRNA production. Example sequences are presented for the CRISPR array of Clostridium thermocellum. Click here to view larger figure.
Figure 3. Cas6 endonuclease assay. A.) Toluidine blue stained polyacrylamide gel of a custom-designed RNA oligonucleotide (250 pmol) and two in vitro RNA transcripts (5 μl of a typical 100 μl reaction). B.) SDS-PAGE gel of a Cas6 preparation (80 pmol) from Clostridium thermocellum after heat precipitation at 50°C for 1 hour and Ni-NTA chromatography. C.) Detection of endonucleolytic Cas6 activity for 5' terminal labeled repeat sequences and pre-crRNA in vitro transcripts. The introduction of a dNTP at position -9 abolishes Cas6 cleavage for a short Cas RNA substrate (S, Fig. 2C). A 5' terminal 8 nt tag is also generated for crRNA maturation from long pre-crRNA substrates (L, Fig. 2A). The bands were separated on a denaturing 8 M urea 12% polyacrylamide gel and visualized by autoradiography.
The presented methods enable the generation of Cas endonuclease substrates of different size ranges and with varying freedom in sequence design. The most straight-forward approach for the generation of synthetic RNA oligonucleotide substrates is limited to short RNA designs due to increasing costs and technical limitations in creating longer RNA oligomers. While successful RNA synthesis has been reported for unmodified RNA oligomers of over 100 nucleotides length, the practical and economical maximum for custom RNA synthesis lies below 40 nucleotides. However, any given sequence can be synthesized and the targeted introduction of modified nucleotides (e.g. deoxyribonucleotides) can be utilized to analyze cleavage sites in detail. Longer pre-cRNAs should be generated via in vitro transcription.
The annealing of oligonucleotides that contain a T7 RNA polymerase promoter allows for the production of intermediate length pre-crRNAs. The maximum length of routinely and economically synthesized DNA oligonucleotides is just above 150 nucleotides and represents the maximum of synthetic pre-crRNAs via this method. The assembly of several annealed DNA oligonucleotide pairs that form sticky ends with each other can extend this maximal length but necessitates increasing challenges in the cloning of the construct. The main advantage of this method is the capability to generate pre-crRNA constructs (and therefore Cas endonuclease substrates) with any desired sequence. This allows the testing of synthetic crRNA designs.
Finally, larger pre-crRNAs can be obtained from PCR amplificates of entire genomic CRISPR elements or fractions thereof. Alterations to the in vitro transcription template plasmid can be introduced via site-directed mutagenesis for the generation of pre-crRNA variants. These constructs can be used to analyze the global endonucleolytic cleavage pattern within an entire pre-crRNA.
The pioneer work for the production of unmodified RNA via T7 RNA polymerase in vitro transcription was based on transfer RNAs 14 and brome mosaic virus RNA 15. The consensus T7 RNA polymerase promoter consists of a recognition domain (-17 through -5) and an initiation domain (-4 through +6) with transcription initiation at an essential guanosine +1 16. Positions downstream of +1 can be varied which allows for the transcription of almost any desired RNA sequence. The presented methods for generating in vitro run-off transcription templates allow for in vitro synthesis of complete pre-crRNAs that match genomic CRISPR regions or synthetic pre-crRNA variants. The transcripts are generated with a 5'-terminal triphosphate present unless the transcription reaction is primed with GMP to obtain 5' monophosphate termini 14. Such termini are required if the transcripts are to be labeled with T4 polynucleotide kinase and γ-[32P]-ATP. Cleavage activity of Cas6 and Cas6-like enzymes generates crRNA that contain 5'-hydroxyl and a 2',3'-cyclic phosphate termini. These RNAs can then be analyzed for recognition by the Cascade complex.
No conflicts of interest declared.
The authors would like to thank Jeanette Schermuly for the preparation of recombinant T7 RNA polymerase and Norman Ebelt for assistance in preparing Figure 1. This work was funded by funds from the Deutsche Forschungsgemeinschaft (DFG FOR1680) and the Max Planck Society.