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The rational design of igRNA is adding additional sequences in the 5' of sgRNA. <br>
 
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Guide RNA (gRNA) is a piece of RNAs that function as guides for RNA- or DNA-targeting enzymes, which they form complexes with. Very often these enzymes will delete, insert or otherwise alter the targeted DNA. <br>
 
 
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<p style="text-align: center;"> Figure 1. Schematic of the design procedure of the gRNA construction and screening. There are five 20nt sequences to change into five different gRNAs.</p>
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<p style="text-align: center;"> Figure 1. Schematic of gRNA and igRNA.</p>
 
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In the CRISPR/Cas9 system, gRNA can direct Cas9 protein to cut dsDNA at the sequence target site. Simultaneously, a hybridization region of igRNA is adding additional sequences in the 5' of sgRNA. Therefore, designing an active gRNA with high cleavage ability is important.<br>
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The “igRNA” stands for RNA-interacting guide RNA, which switch to their active state only upon interaction with target RNAs (Figure 1).<br>
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Based on the target gene(tetR), we chose CGG and AGG as PAM, and designed 5 gRNAs in the plasmid pTargetF (Figure 1). <br>
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The additional sequences include recognition sequence A, cis-repressed sequence and recognition sequence B. The cis-repressed sequence is complementary to the spacer, leading to the repression of the binding ability between igRNA with its DNA target. The recognition sequence A and the recognition sequence B are complementary to the interacting sequence A and interacting sequence B, respectively.<br>
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All designed igRNAs were analyzed using <a href="http://www.nupack.org/">NUPACK webserver</a>.<br>
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The igRNA that has lower free energy, co-folding energy with target RNA as well as reduced self-comlementarity at 29℃ would be the best (Figure 2).
 
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Latest revision as of 14:32, 20 October 2021


Interacting Guide RNA（igRNA）

The rational design of igRNA is adding additional sequences in the 5' of sgRNA.

Usage and Biology

Figure 1. Schematic of gRNA and igRNA.

The “igRNA” stands for RNA-interacting guide RNA, which switch to their active state only upon interaction with target RNAs (Figure 1).
The additional sequences include recognition sequence A, cis-repressed sequence and recognition sequence B. The cis-repressed sequence is complementary to the spacer, leading to the repression of the binding ability between igRNA with its DNA target. The recognition sequence A and the recognition sequence B are complementary to the interacting sequence A and interacting sequence B, respectively.

All designed igRNAs were analyzed using NUPACK webserver.
The igRNA that has lower free energy, co-folding energy with target RNA as well as reduced self-comlementarity at 29℃ would be the best (Figure 2).


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]