Difference between revisions of "Part:BBa K4223003"
 
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<partinfo>BBa_K4223003 short</partinfo>
 
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Cas13a tolerates one or two mismatches between crRNA and the target sequence, which will result in a greatly reduced cleavage efficiency of Cas13a protein, and this wrong cleavage will also lead to "false positives" in the detection. <br>
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Cas13a tolerates one or two mismatches between sgRNA and the target sequence, which will result in a greatly reduced cleavage efficiency of Cas14a protein, and this wrong cleavage will also lead to "false positives" in the detection. <br>
 
In order to confirm the occurrence of false positives, Hainanu-China designed a series of sequences with single nucleotide polymorphisms (SNPS) based on the target RNA, which were detected by fluorescence reporter. <br>
 
In order to confirm the occurrence of false positives, Hainanu-China designed a series of sequences with single nucleotide polymorphisms (SNPS) based on the target RNA, which were detected by fluorescence reporter. <br>
 
It should be noted that fluorescence was generated by trans-cleavage of ssRNA cleavage Reporter after Cas13a recognized and cleaved the target RNA.
 
It should be noted that fluorescence was generated by trans-cleavage of ssRNA cleavage Reporter after Cas13a recognized and cleaved the target RNA.
 
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<h3>Description</h3>
 
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Single-stranded DNA-G-quadruplex is used as the template of the RCA, which amplifies G-quadruplex sequence. G-quadruplex can form a specific secondary topological structure after annealing, which can work as a DNAenzyme through Hoogsten bond association with hemin. This kind of DNA mimic enzyme can catalyze hydrogen peroxide reaction to form O<sup>2-</sup> ion. The reaction between the substrate ABTS solution and hydrogen peroxide was used as the background. After the addition of G-quadruplex/hemin mimic enzyme, ABTS quickly reacted with O<sup>2-</sup> ions to form ABTS<sup>-</sup> ions, which changed the color of the system from colorless to green<sup>[1]</sup>. Therefore, we use a DNA-based system that activates a RCA process after the G-quadruplex-containing DNA is recognized by the ssDNA replaced by Phi29. The RCA process will subsequently produce DNA chains composed of numerous G-quadruplex units. After skipping the primer removal and recombination processes, we directly put the primer-template hybrid chain mix into the RCA, which simplifies the experimental procedure and saves the cost of exonuclease to specifically remove ssDNA.
 
 
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<h3>Experience</h3>
 
<h3>Experience</h3>
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The iGEM 2021 NEFU_China introduced the single stand sequence of G-quadruplex with ssDNA into the circular DNA template. This was achieved by primer incubation, followed by the addition of Phi29 DNA polymerase and dNTPs into the RCA buffer; hemin was then added into the reaction system followed by incubation and ABTS addition. With the presence of the viral RNA sequence in the initial sample, the G-quadruplex will be amplified and the reaction solution will change to green color<sup>[1-2]</sup>.
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'''Cas13a system'''<br>
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In order to provide data support for false positives, we introduced 13 RNA sequences into Cas13a detection system, including 1 Target RNA and 12 RNA mutated sequences containing odd locus single base mutations. The specific mutation sites are shown in Figure 1.
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<img src="https://2021.igem.org/wiki/images/c/c1/T--NEFU_China--NEFU-xiguangdu.png" style="width:40%;height=40%; "></img>
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Figure 1. Absorbance curves of different concentrations of G-quadruplex.
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In order to put our detection device into actual application, we explored the relationship between absorbance and the primer amount according to the detectable color range, and finally determined the required sample size through the modeling. Therefore, we designed a gradient of primer concentrations for RCA and chromogenic reaction, and obtained the results.
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<img src="https://2021.igem.org/wiki/images/7/75/T--NEFU_China--RCA_con.png" style="width:30%;height=30%; "></img>
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Figure 2. Results of G-quadruplex electrophoresis by Phi29-mediated RCA.
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<img src="https://2021.igem.org/wiki/images/4/42/T--NEFU_China--Results-21.png" style="width:45%;height=60%; "></img>
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Figure 3. Chromogenic reaction of Phi29-mediated RCA products. | 1. Positive control with a synthesized G-quadruplex sequence. 2. Products of Phi29-mediated RCA. 3. Reaction without primers. 4. Reaction without template. 5. Reaction without Phi29 polymerase.  6. Reaction without T4 DNA ligase.
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<img src="https://2021.igem.org/wiki/images/d/db/T--NEFU_China--Results-22.png" style="width:45%;height=45%; "></img>
 
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Figure 4. Absorbance at 420nm of the chromogenic reaction of the Phi29-mediiated RCA products.
 
 
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[[File:Single nucleotide polymorphisms(SNPS) at odd loci in 12 different RNA and Target RNA.png|500px|]]<br>
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'''Figure 1. Single nucleotide polymorphisms(SNPS) at odd loci in 12 different RNA and Target RNA'''
 
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The iGEM 2021 NEFU_China used purified Phi29 polymerase to carry out RCA for the amplification the G-quadruplex template. Based on the DNA agarose electrophoresis, the RCA products could be steadily detected in the experimental group but not in the controls (Figure 2). Meanwhile, the color change could also be observed in the experimental group versus the controls (Figure 3), and their absorbance at 420 nm was also quantified (Figure 4).  From the results of these three experiments, we conclude that Phi29 can efficiently amplify the G-quadruplex sequence through the RCA reaction and the amplified product can generate chromogenic reaction.
 
 
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Through the recognition and cleavage of the artificially designed RNA by Cas13a protein, the trans-cleavage activity was activated, and the resulting fluorescence signal was revealed as Relative fluorescence Unit (RFU). We could observe that each detected sequence showed different fluorescence signal intensities, and the signal intensities of RM8 RM15 and RM19 were significantly greater than the target RNA. This indicates that false positives do exist, and different mutation sites have different effects on the specificity of the detection.
 
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<h3>References</h3>
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[[File:Detection results of Cas13a protein for RNA sequences (revealed by relative fluorescence unit RFU).png|400px|]]<br>
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[1] R. Connelly, C. Verduzco, et al., Toward a rational approach to design split G-quadruplex probes, ACS Chem. Bio, 10:1021, 2019.
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'''Figure 2. Detection results of Cas13a protein for each sequence (revealed by relative fluorescence unit RFU)'''<br>
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[2] T. Yoshimura, S. Arikado and S. Ohuchi. Estimation of DNA polymerase for improvement of rolling circle amplification, Oxford University Press, 820-8502, 2006.
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<span class='h3bb'>Sequence and Features</span>
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<h3>Centrifuge tube system</h3>
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(2x) buffer: 20 mM tris-hcl, KCl 100 mM, pH=8.2.<br><br>
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20 uL /cell：<br><br>
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10uL  buffer(2x);<br>
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0.8uL  500nM Cas13a;<br>
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0.1uL  5uM crRNA;<br>
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0.67uL 150mM Mg2+(ca. 5mM Mg2+ final.) <br><br>
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37 ℃ Incubation 10-15min, add probe 0.6uL（10uM） FQ ;<br>
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（12.17 uL in total  ca. 12 uL)
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Latest revision as of 13:47, 21 September 2022


Transcription system of target RNA for LbCas13a

Cas13a tolerates one or two mismatches between sgRNA and the target sequence, which will result in a greatly reduced cleavage efficiency of Cas14a protein, and this wrong cleavage will also lead to "false positives" in the detection.
In order to confirm the occurrence of false positives, Hainanu-China designed a series of sequences with single nucleotide polymorphisms (SNPS) based on the target RNA, which were detected by fluorescence reporter.
It should be noted that fluorescence was generated by trans-cleavage of ssRNA cleavage Reporter after Cas13a recognized and cleaved the target RNA.


Experience

Cas13a system
In order to provide data support for false positives, we introduced 13 RNA sequences into Cas13a detection system, including 1 Target RNA and 12 RNA mutated sequences containing odd locus single base mutations. The specific mutation sites are shown in Figure 1.

Single nucleotide polymorphisms(SNPS) at odd loci in 12 different RNA and Target RNA.png
Figure 1. Single nucleotide polymorphisms(SNPS) at odd loci in 12 different RNA and Target RNA

Through the recognition and cleavage of the artificially designed RNA by Cas13a protein, the trans-cleavage activity was activated, and the resulting fluorescence signal was revealed as Relative fluorescence Unit (RFU). We could observe that each detected sequence showed different fluorescence signal intensities, and the signal intensities of RM8 RM15 and RM19 were significantly greater than the target RNA. This indicates that false positives do exist, and different mutation sites have different effects on the specificity of the detection.
Detection results of Cas13a protein for RNA sequences (revealed by relative fluorescence unit RFU).png
Figure 2. Detection results of Cas13a protein for each sequence (revealed by relative fluorescence unit RFU)





Centrifuge tube system

(2x) buffer: 20 mM tris-hcl, KCl 100 mM, pH=8.2.

20 uL /cell：

10uL buffer(2x);
0.8uL 500nM Cas13a;
0.1uL 5uM crRNA;
0.67uL 150mM Mg2+(ca. 5mM Mg2+ final.)

37 ℃ Incubation 10-15min, add probe 0.6uL（10uM） FQ ;
（12.17 uL in total ca. 12 uL)



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 100
  • 1000
    COMPATIBLE WITH RFC[1000]