Difference between revisions of "Part:BBa K4586004"

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==Part Description==
 
==Part Description==
  
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This part codes for the type V-K CRISPR-Cas system effector protein Cas12k, that is characterized by naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA or genome editing, Its protospacer adjacent motif (PAM) sequence is GGTT. that is essential for activating the enzymatic activity of the Cas protein, and the guide RNA is designed based on the PAM sequence site corresponding to the target gene.  
+
We have added this novel part that codes for the type V-K CRISPR-Cas system effector protein Cas12k, that is characterized by naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA or genome editing, Its protospacer adjacent motif (PAM) sequence is GGTT. that is essential for activating the enzymatic activity of the Cas protein, and the guide RNA is designed based on the PAM sequence site corresponding to the target gene.
  
 
==Usage==
 
==Usage==
 
This part is implemented in our design to be the main element of our therapeutic agent (Cargo) to knock-out the BAFF-R gene apoptosis, which is essential for B-cell survival and maintenance, to cause apoptosis in the autoreactive B-cells that secrete Anti-citrullinated Protein Antibody (ACPA). Its guide RNA is designed to drive the protein portion of the CRISPR Cas system Cas12k to the BAFF-R gene as shown in figure 1.
 
This part is implemented in our design to be the main element of our therapeutic agent (Cargo) to knock-out the BAFF-R gene apoptosis, which is essential for B-cell survival and maintenance, to cause apoptosis in the autoreactive B-cells that secrete Anti-citrullinated Protein Antibody (ACPA). Its guide RNA is designed to drive the protein portion of the CRISPR Cas system Cas12k to the BAFF-R gene as shown in figure 1.
 +
We used this specific type of CRISPR-Cas system as it's considered one of the shortest forms of Cas proteins. Therefore, Cas12k fits into the limited loading capacity of our engineered exosomes.
 
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 
width:100%;
 
width:100%;
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">
 
">
 
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'Figure 1: This figure illustrates the activity of the Cas12k protein into the target autoreactive B-cells through knocking down the B-cell activating factor receptor (BAFF-R) gene that is responsible for B-cells survival and proliferation leading to their apoptosis.
+
lang=EN style='font-size:11.0pt;line-height:115%'>Figure 1: This figure illustrates the activity of the Cas12k protein into the target autoreactive B-cells through knocking down the B-cell activating factor receptor (BAFF-R) gene that is responsible for B-cells survival and proliferation leading to their apoptosis.
 
   </span></p></div></html>
 
   </span></p></div></html>
  
 +
==Literature Characterization==
 +
In order to purify Cas12k,  it was expressed in Escherichia coli.Cas12k's capacity to catalyze DNA transposition was tested using gel electrophoresis. To ascertain the background level of transposition,  a negative control response devoid of Cas12k was used.
 +
<html><div align="center"style="border:solid #17252A; width:50%;float:center;"><img style="                              max-width:850px;
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width:75%;
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height:auto;
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position: relative;
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top: 50%;
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left: 35%;
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transform: translate( -50%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/literature-characterisation-parts/cas12k.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 +
lang=EN style='font-size:11.0pt;line-height:115%'>The graph displays the average and standard deviation of three independent replicates from a representative experiment. The level of DNA transposition was markedly elevated by the addition of Cas12k.
 +
</span></p></div></html>
 +
==Characterization By Mutational Landscape==
 +
In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness.  As displayed in the chart below, the mutation (A8C,F13S) shows the highest epistatic fitness, while the lowest score was associated with the mutation (R536E).
 +
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
 +
width:100%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 50%;
 +
transform: translate( -50%);
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padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/parts-de/cas12k.png">
 +
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 +
lang=EN style='font-size:11.0pt;line-height:115%'>Figure . An illustration of the effects of different mutations on the Epistatic Fitness of cas12K.
 +
</span></p></div></html>
 +
==Experimental Characterization==
 +
In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents lane (p9) including second part of MS2, the sensor and cas12k , and then we measured the specific concentration of the running part using Real-Time PCR as shown in the following figure.
 +
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
 +
width:100%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 50%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/pcr-ampli.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>
 +
 +
</span></p></div></html>
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<br><br><br><br>
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We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane(P9).
 +
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
 +
width:100%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 50%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/digestion-2.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>
 +
 +
</span></p></div></html>
 +
<br><br><br><br>
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After the ligation step, we cultured the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the pCDNA3(-) plasmid vector containing insert parts.
 +
 +
 +
 +
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
 +
width:100%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 50%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/results/3.png">
 +
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 +
lang=EN style='font-size:11.0pt;line-height:115%'>
 +
 +
</span></p></div></html>
  
 +
==References==
 +
Xiao, R., Wang, S., Han, R., Li, Z., Gabel, C., Mukherjee, I. A., & Chang, L. (2021). Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition. Molecular Cell, 81(21), 4457-4466.
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 15:48, 12 October 2023


Cas12k

Part Description

We have added this novel part that codes for the type V-K CRISPR-Cas system effector protein Cas12k, that is characterized by naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA or genome editing, Its protospacer adjacent motif (PAM) sequence is GGTT. that is essential for activating the enzymatic activity of the Cas protein, and the guide RNA is designed based on the PAM sequence site corresponding to the target gene.

Usage

This part is implemented in our design to be the main element of our therapeutic agent (Cargo) to knock-out the BAFF-R gene apoptosis, which is essential for B-cell survival and maintenance, to cause apoptosis in the autoreactive B-cells that secrete Anti-citrullinated Protein Antibody (ACPA). Its guide RNA is designed to drive the protein portion of the CRISPR Cas system Cas12k to the BAFF-R gene as shown in figure 1. We used this specific type of CRISPR-Cas system as it's considered one of the shortest forms of Cas proteins. Therefore, Cas12k fits into the limited loading capacity of our engineered exosomes.

Figure 1: This figure illustrates the activity of the Cas12k protein into the target autoreactive B-cells through knocking down the B-cell activating factor receptor (BAFF-R) gene that is responsible for B-cells survival and proliferation leading to their apoptosis.

Literature Characterization

In order to purify Cas12k, it was expressed in Escherichia coli.Cas12k's capacity to catalyze DNA transposition was tested using gel electrophoresis. To ascertain the background level of transposition, a negative control response devoid of Cas12k was used.

The graph displays the average and standard deviation of three independent replicates from a representative experiment. The level of DNA transposition was markedly elevated by the addition of Cas12k.

Characterization By Mutational Landscape

In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness. As displayed in the chart below, the mutation (A8C,F13S) shows the highest epistatic fitness, while the lowest score was associated with the mutation (R536E).

Figure . An illustration of the effects of different mutations on the Epistatic Fitness of cas12K.

Experimental Characterization

In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents lane (p9) including second part of MS2, the sensor and cas12k , and then we measured the specific concentration of the running part using Real-Time PCR as shown in the following figure.





We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane(P9).





After the ligation step, we cultured the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the pCDNA3(-) plasmid vector containing insert parts.


References

Xiao, R., Wang, S., Han, R., Li, Z., Gabel, C., Mukherjee, I. A., & Chang, L. (2021). Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition. Molecular Cell, 81(21), 4457-4466. 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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]