Substrate Generation for Endonucleases of CRISPR/Cas Systems

1. Generation of Long Pre-crRNA Substrates via PCR

1. Design PCR primers targeting the spacer regions of a CRISPR cluster. Add the T7 RNA polymerase (T7RNAP) promoter sequence (5′ -taatacgactcactata-3′) to the forward primer and restriction sites for cloning the PCR product into a vector to both primers (e.g. BamHI and Hind III for pUC19, Fig. 2A).
   Note: The T7RNAP requires a guanidine residue for proper initiation of transcription.
2. Amplify your pre-crRNA sequence of interest from genomic DNA by PCR.
3. Separate the PCR products by agarose gel electrophoresis and gel extract the desired band. Digest the PCR product with the restriction enzymes to create sticky ends (e.g. BamHI and HindIII, Fig. 2A). Purify your PCR product with a PCR purification kit to eliminate cleavage by-products.
4. Set up a ligation reaction that contains T4 DNA ligase, T4 DNA ligase buffer and a 3:1 molar ratio of the cleaved PCR product and the dephosphorylated linear pUC vector with corresponding sticky ends. Incubate the mixture at 16 °C overnight. Transform the ligation mixture into competent Escherichia coli DH5α cells by standard protocols and use blue white screening to identify successful ligation.
5. Isolate plasmids from white colonies using a plasmid preparation kit. Identify positive clones by plasmid sequencing. Alternatively, colony PCR might be utilized for screening.

2. Generation of Intermediate Pre-crRNA Substrates via Annealing of DNA Oligonucleotides

1. Design forward and reverse oligonucleotides with the desired CRISPR repeat/spacer sequence. The oligonucleotides contain the sequence of a T7 RNAP promoter as well as terminal restriction sites (e.g. BamHI and Hind III for pUC19). Terminate the oligonucleotides to ensure that sticky ends form after annealing (see Fig. 2B).
2. 5′-phosphorylate 1 nmol of each oligonucleotide in a separate 20μl reaction containing 5 μl of T4 polynucleotide kinase (PNK), 2 μl of T4 PNK 10x buffer, 2 μl ATP (10 mM). Incubate each sample for 1 hour at 37 °C.
3. Hybridize the two phosphorylated oligonucleotides. Combine 1μl of the phosphorylated forward oligo mixture (from 2.2.), 1 μl of the phosphorylated reverse oligo mixture (from 2.2.), 1 μl of T4 DNA ligase 10x buffer in a 10 μl reaction. Incubate the samples for 5 min at 95 °C on a heating block or in boiling water, turn off the heat source and let the mixture cool down to room temperature (~2-3 hours).
   Note: In this critical step, the slow cooling process favors the annealing of the two oligonucleotides compared to the formation of structures within each single oligonucleotide.
4. Ligate 4 μl of the hybridization mix, 1μl of digested and dephosphorylated pUC vector (0.1 μg/μl) with T4 DNA ligase, T4 DNA ligase 10x buffer and 10 mM ATP in a 20 μl ligation mixture. Incubate the sample at 16 °C overnight.
5. Transform the ligated plasmids into competent Escherichia coli DH5α cells by standard protocols and utilize blue white screening. Isolate plasmids and identify positive clones by digestion (to screen for inserts of the desired size) and subsequent plasmid sequencing.

3. Generation of Short Cas RNA Substrates via Custom RNA Oligonucleotide Synthesis

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.

4. In vitro T7 RNA Polymerase Transcription

1. Isolate plasmids with your designed construct (from 1.9. or 2.7.) using a maxiprep plasmid purification kit.
2. Linearize the plasmid with the restriction enzyme that cleaves downstream of the cloned fragment (e.g. HindIII). Ensure complete digestion.
   Note: If a divergent defined 3′ terminus of the RNA transcript is desired, the designed construct should contain an additional specific restriction site for “run-off” transcription upstream of the HindIII sequence.
3. Purify the linearized plasmid by phenol:chloroform (1:1) extraction and ethanol precipitation. Recover the nucleic acids by resuspending the pellet in DEPC treated sterile water.
4. Set up an in vitro T7 RNAP run off transcription mixture that contains 40 mM Hepes/KOH (pH 8), 22 mM MgCl₂, 5 mM dithiothreitol, 1 mM spermidine, 4 mM of each nucleoside triphosphate (ATP, CTP, GTP, UTP), 40-100 μg/ml of digested plasmid and 0.1 mg/ml T7 RNAP in DEPC treated water. Incubate for 3 hours at 37 °C.
5. Analyze the obtained RNA transcripts on a denaturing 8 M urea 12% polyacrylamide gel (Fig. 3A). The RNA transcripts can be purified via Mono Q anion exchange chromatography¹³ and recovered by ethanol precipitation of the RNA fractions and resuspension of the pellet in DEPC treated sterile water. For future use, store the RNA at -80 °C.

5. Cas6 Endonuclease Assay

1. Set up a 20 μl in vitro T7 RNAP run off transcription mixture (see 4.4) that contains a reduced amount of 2 mM ATP and is complemented with 2.5 μl α-[³²P]-ATP (10 mCi/ml, 5000 Ci/mmol). Purify reaction products via gel extraction from a denaturing 8 M urea 12% polyacrylamide gel. Visualize bands by autoradiography.
2. Produce and purify the desired recombinant Cas proteins. In this example, Cas6 from Clostridium thermocellum was purified via heat precipitation and Ni-NTA chromatography.
3. Set up an endonuclease assay reaction (e.g. for Clostridium thermocellum Cas6, the reaction mixture contains 20 mM Hepes (KOH pH8), 250 mM KCl, 2 mM MgCl₂, 1 mM DTT, 12,000 cpm RNA substrate and 1 μM enzyme and was incubated at 37 °C for 30 min).
4. Load 5 μl of the reaction mixture (+ 10 μl RNA loading buffer containing 95% formamide) on a 8 M urea 12% polyacrylamide gel. Visualize the cleavage products after electrophoresis by autoradiography.

6. Representative Results

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.