Difference between revisions of "Part:BBa K3791015"
 
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<partinfo>BBa_K3791015 short</partinfo>
 
<partinfo>BBa_K3791015 short</partinfo>
  
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This part is an improved version of the ampicillin gRNA: [https://parts.igem.org/Part:BBa_K3791005 BBa_K3791005]. Thus, its function is the same as the one mentioned there: to target a specific ampicillin-resistance gene fragment. However, its efficiency was increased by using the architecture that (will be now further explained) is going to be presented afterwards. Our idea originated when becoming aware of Cas12a ability to process its own CRISPR RNAs (crRNAs). This means that, once the succession of direct repeat (DR) and spacer sequences are transcribed, the resulting transcript (named pre-crRNA) can be processed into mature gRNAs as a result of the dual RNase/DNase activity of Cas12a [1]. For that event to happen, the spacer sequence (necessary for target DNA recognition) should be followed and preceded by a DR. Specifically, it is known that Cas12 cuts the pre-crRNA 4 nucleotides upstream of the hairpin structures formed by the DR [2]. That was taken into account when designing all these efficient gRNAs ([https://parts.igem.org/Part:BBa_K3791016 BBa_K3791016], [https://parts.igem.org/Part:BBa_K3791017 BBa_K3791017], [https://parts.igem.org/Part:BBa_K3791018 BBa_K3791018] and [https://parts.igem.org/Part:BBa_K3791019 BBa_K3791019]), in order not to lose key nucleotides after Cas12a processing. From this knowledge, the following sequence structure emerged: <b>repeat + spacer + 4 nucleotides +  repeat</b>.
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This part is an improved version of the ampicillin gRNA: <html><a href="https://parts.igem.org/Part:BBa_K3791005" target="_blank" onmouseover="this.style.color='#ff724a';" onmouseout="this.style.color='#ff9515';" style="color: #ff9515; text-decoration: none;">BBa_K3791005</a></html>. Thus, its function is the same as the one mentioned there: to target a specific ampicillin-resistance gene fragment. However, its efficiency was increased by using the architecture that will be now further explained. Our idea originated when becoming aware of Cas12a ability to process its own CRISPR RNAs (crRNAs). This means that, once the succession of direct repeat (DR) and spacer sequences are transcribed, the resulting transcript (named pre-crRNA) can be processed into mature gRNAs as a result of the dual RNase/DNase activity of Cas12a [1]. For that event to happen, the spacer sequence (necessary for target DNA recognition) should be followed and preceded by a DR. Specifically, it is known that Cas12 cuts the pre-crRNA 4 nucleotides upstream of the hairpin structures formed by the DR [2]. That was taken into account when designing all these efficient gRNAs (<html><a href="https://parts.igem.org/Part:BBa_K3791016" target="_blank" onmouseover="this.style.color='#ff724a';" onmouseout="this.style.color='#ff9515';" style="color: #ff9515; text-decoration: none;">BBa_K3791016</a></html>, <html><a href="https://parts.igem.org/Part:BBa_K3791017" target="_blank" onmouseover="this.style.color='#ff724a';" onmouseout="this.style.color='#ff9515';" style="color: #ff9515; text-decoration: none;">BBa_K3791017</a></html>, <html><a href="https://parts.igem.org/Part:BBa_K3791018" target="_blank" onmouseover="this.style.color='#ff724a';" onmouseout="this.style.color='#ff9515';" style="color: #ff9515; text-decoration: none;">BBa_K3791018</a></html> and <html><a href="https://parts.igem.org/Part:BBa_K3791019" target="_blank" onmouseover="this.style.color='#ff724a';" onmouseout="this.style.color='#ff9515';" style="color: #ff9515; text-decoration: none;">BBa_K3791019</a></html>), in order not to lose key nucleotides after Cas12a processing. From this knowledge, the following sequence structure emerged: <b>repeat + spacer + 4 nucleotides +  repeat</b>.
  
  
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===Usage, Biology and Characterization===
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===Usage and Biology===
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As this part is a simplification of our final construct, usage, biology and characterization is explained there: <html><a href="https://parts.igem.org/Part:BBa_K3791020" target="_blank" onmouseover="this.style.color='#ff724a';" onmouseout="this.style.color='#ff9515';" style="color: #ff9515; text-decoration: none;">BBa_K3791020</a></html>
  
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'><b>Sequence and Features</b></span>
 
<partinfo>BBa_K3791015 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3791015 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K3791015 parameters</partinfo>
 
<partinfo>BBa_K3791015 parameters</partinfo>
 
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===References===
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[1] Campa, C. C., Weisbach, N. R., Santinha, A. J., Incarnato, D., & Platt, R. J. (2019). <b>Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts.</b> <i>Nature Methods, 16</i>(9), 887–893. <html><a href="https://doi.org/10.1038/s41592-019-0508-6" target="_blank" onmouseover="this.style.color='#ff724a';" onmouseout="this.style.color='#ff9515';" style="color: #ff9515; text-decoration: none;">https://doi.org/10.1038/s41592-019-0508-6</a></html>
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<br> [2] Paul, B., & Montoya, G. (2020). <b>CRISPR-Cas12a: Functional overview and applications.</b> <i>Biomedical Journal, 43</i>(1), 8–17. <html><a href="https://doi.org/10.1016/j.bj.2019.10.005" target="_blank" onmouseover="this.style.color='#ff724a';" onmouseout="this.style.color='#ff9515';" style="color: #ff9515; text-decoration: none;">https://doi.org/10.1016/j.bj.2019.10.005</a></html>

Latest revision as of 21:09, 21 October 2021


Efficient gRNA Ampicillin

This part is an improved version of the ampicillin gRNA: BBa_K3791005. Thus, its function is the same as the one mentioned there: to target a specific ampicillin-resistance gene fragment. However, its efficiency was increased by using the architecture that will be now further explained. Our idea originated when becoming aware of Cas12a ability to process its own CRISPR RNAs (crRNAs). This means that, once the succession of direct repeat (DR) and spacer sequences are transcribed, the resulting transcript (named pre-crRNA) can be processed into mature gRNAs as a result of the dual RNase/DNase activity of Cas12a [1]. For that event to happen, the spacer sequence (necessary for target DNA recognition) should be followed and preceded by a DR. Specifically, it is known that Cas12 cuts the pre-crRNA 4 nucleotides upstream of the hairpin structures formed by the DR [2]. That was taken into account when designing all these efficient gRNAs (BBa_K3791016, BBa_K3791017, BBa_K3791018 and BBa_K3791019), in order not to lose key nucleotides after Cas12a processing. From this knowledge, the following sequence structure emerged: repeat + spacer + 4 nucleotides + repeat.


Usage, Biology and Characterization

As this part is a simplification of our final construct, usage, biology and characterization is explained there: BBa_K3791020


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]


References

[1] Campa, C. C., Weisbach, N. R., Santinha, A. J., Incarnato, D., & Platt, R. J. (2019). Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nature Methods, 16(9), 887–893. https://doi.org/10.1038/s41592-019-0508-6
[2] Paul, B., & Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications. Biomedical Journal, 43(1), 8–17. https://doi.org/10.1016/j.bj.2019.10.005