The overall goal of the following experiments is to generate RNA substrates for the study of Cas Endonuclease, which play crucial roles in the crispr Cass immune systems found in bacteria and ArcHa to obtain RNA substrates for Cas Endonuclease. PCR amplification of a specific CRISPR region can be employed to obtain long DNA template for in vitro RNA production. If preferred.
Two oligonucleotides can be a kneeled to generate shorter templates for substrate RNA production. Alternatively, short, custom synthesized repeat sequences can then be tested in CAS Endonuclease assays. Ultimately, the production of RNA transcripts and the subsequent cleavage by CAS six endonuclease can be visualized by auto radiography.
The main goal of the presenter techniques is to enable researchers to study CRISPR RNA processing with RNA substrates of varying length. Therefore, different methods are utilized to generate small RNAs, for example, single repeat elements or larger RNAs such as crispr, RNA, transcripts that span several spacers. Though these methods specifically provide insight into CRISPR RNA maturation similar template preparation strategies can be adopted for other RNA processing systems, Demonstrating the procedure will be muted surface a graduate student and repl the postdoc, both from a laboratory In bacteria and ArcHa CRISPR immune systems.
A viral DNA sequence called a proto spacer can be inserted between two repeat elements in a growing CRISPR cluster in a process called adaptation. Next, the entire CRISPR cluster is transcribed and then processed into small CRISPR RNAs by a Cass Endonuclease, for example, CAS six. The small CRISPR RNAs are then incorporated into the CAS protein complex cascade and mediate the host defense against a repeated viral attack, utilizing the base complementarity between the CRISPR RNA and the proto spacer as a signal for viral DNA degradation to generate long pre CRISPR RNA substrates.
Begin by designing PCR primers, targeting the spacer regions of a CRISPR cluster by adding the T seven RNA polymerase promoter sequence to the forward primer. Then add restriction sites to both primers for cloning the PCR product into a vector. Note that the T seven RNA polymerase requires I guine residue for proper initiation of transcription after amplifying the pre CRISPR RNA sequence of interest from the genomic DNA by PCR.
Separate the PCR products, Viagra aose gel electropheresis and gel extract the desire banned, then digest the PCR product with restriction enzymes to create sticky ends. Next, after purifying your PCR product with a PCR purification kit, to eliminate any cleavage byproducts, add T four DNA Ligase T four DNA Ligase buffer, and a three to one molar ratio of the cleaved PCR product to the DFOs four related puck vector with the corresponding sticky ends. Now incubate the ligation reaction at 16 degrees Celsius overnight, and then transform the mixture into competent e coli, DH five alpha cells by standard protocols using blue white screening to identify successful ligation.
Finally, isolate the plasmids from the white colonies using a plasmid preparation kit and identify positive clones by plasmid sequencing to generate intermediate sized pre CRISPR RNA substrates. Begin by designing forward and reverse oligonucleotides with the desired CRISPR repeat spaces sequence similar to the sequences shown here. Note that the oligonucleotides contain the sequence of a T seven RNA polymerase promoter as well as terminal restriction sites after terminating the oligonucleotides to ensure that sticky ends form.
After a kneeling incubate the samples with T four polynucleotide kinase and T four polynucleotide kinase buffer for one hour at 37 degrees Celsius to five prime phosphorylate, one anomal of each of the oligonucleotides. Then incubate the phosphorylated forward and reverse oligonucleotides with T four DNA ligase buffer for five minutes at 95 degrees Celsius on a heating block. After the samples have hybridized, turn off the heat source and let the mixture cool down for about two to three hours until they reach room temperature.
Now ligate the called oligonucleotides with a hybridization mix digested and dephosphorylated P vector T four DNA Ligase buffer, and a TP at 16 degrees Celsius overnight. Then transform the ligated plasmids into competent e coli, DH five alpha cells by standard protocols using blue white screening to identify successful ligation. Finally, isolate the plasmids and identify the positive clones by digestion and subsequent plasmid sequencing.
Short single repeat cas RNA substrate sequences can be designed similar to the ones shown here. Be certain to include a deoxy ribonucleotide. If the site of RNA cleavage was to be determined, a custom synthesis facility can then be utilized to implement the production of the RNA oligonucleotide.
After isolating plasmids with the customized substrate of choice, using a maxi prep plasmid purification kit linearize the plasmids with the restriction enzyme that cleaves downstream of the cloned fragments. Then after ensuring complete digestion, purify the linearized plasmid by phenol chloroform extraction and ethanol precipitation. Recover the nucleic acids by resus, suspending the pellet in DEPC treated sterile water.
Now incubate the digestive plasmids in a freshly prepared in vitro T seven RNA polymerase runoff transcription mixture including A-T-P-C-T-P-G-T-P, and UTP for three hours at 37 degrees Celsius. Finally, analyze the obtained RNA transcripts on a denaturing eight molar urea, 12%poly acrylamide gel. It is also possible to purify the transcripts via mono Q anion exchange chromatography, and then recover the transcripts by ethanol precipitation of the RNA fractions and resus of the pellets in DEPC treated sterile water to analyze the design substrates by endonuclease assays begin by visualizing the substrate bands by auto radiography and purifying the reaction products by gel extraction from a denaturing eight molar urea.
12%poly acrylamide gel then produce and purify the desired recombinant cas proteins via heat precipitation, a nickel NTA chromatography, as shown for this CAS six protein from Clostridium thermo cell. Then combine the CAS protein with a freshly prepared endonuclease reaction mixture for 30 minutes at 37 degrees Celsius to allow the endonuclease assay reaction to occur. Finally, load five of the reaction mixture on an eight molar urea.
12%poly acrylamide gel and visualize the cleavage products after electrophoresis by auto radiography. Here as hine blue stained poly acrylamide gel of a custom designed RNA oligonucleotide and two in vitro RNA transcripts is shown. Note that the efficiency of the RNA production varies between the short, long and intermediate length constructs.
The investigation of RNA endonuclease activity requires positive negative controls, as well as highly purified recombinant cas proteins. In this figure. An SDS page gel of a CAS six sample preparation from Clostridium thermo cell.
After heat precipitation and nickel, NTA chromatography run alongside a standard protein marker is shown. A suitable negative control sample should contain a cell lysate without CAS six expression, but following the identical CAS six purification procedure, the CAS six preparation was determined to possess the required high level of purity. Here, the cleavage of a five prime terminal labeled repeat sequence substrate by co Clostridium thermo cell CAS six protein can be visualized by auto radiography.
Each lane contains radioactively labeled substrate, RNA after incubation with ca six protein as indicated by the plus signs, or with a buffer as a negative control as to noted by the minus signs, long substrates or short substrates with or without the addition of the deoxy ribonucleotide. At position minus nine, were used to facilitate the endonuclease reaction. Note, the lack of cleavage of the short substrates when the deoxy ribonucleotide was included in the RNA molecule Following this procedure.
CAS six Nucleases can also be used as a tool to generate mature, crisp RNAs. These RNAs can be incorporated into cascade complexes to obtain insights into the involvement in the interference of viral attacks. After watching this video, you should have a good understanding of how to design and produce CRISPR es substrates for Cas and nucleases.
These different substrates are useful for addressing questions concerning the maturation and utilization of crispr Es.
