Difference between revisions of "Part:BBa K2818002"
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===Usage and Biology=== | ===Usage and Biology=== | ||
As mentioned above, the dPspCas13b is the catalytically inactive version of Type VI RNA-targeting CRISPR-associated protein 13b, an RNA-guided ribonuclease derived from <html><i>Prevotella sep. P5-125</i> </html>and it acts as the RNA-targeting scaffold to bind to specific RNA target sequence. Such a binding action is mediated by a single guide RNA, the sequence of which greatly affects the binding efficiency of dCas protein onto the target and ultimately the functionality of the fused protein. <br> | As mentioned above, the dPspCas13b is the catalytically inactive version of Type VI RNA-targeting CRISPR-associated protein 13b, an RNA-guided ribonuclease derived from <html><i>Prevotella sep. P5-125</i> </html>and it acts as the RNA-targeting scaffold to bind to specific RNA target sequence. Such a binding action is mediated by a single guide RNA, the sequence of which greatly affects the binding efficiency of dCas protein onto the target and ultimately the functionality of the fused protein. <br> | ||
| − | The other domain fused to the dCas13b here is the Adenosine deaminase acting on RNA 2 (ADAR2), which is an enzyme that catalyzes the hydrolytic deamination of adenosine to inosine. As inosine is functionally equivalent to guanosine, such a construct can be | + | The other domain fused to the dCas13b here is the Adenosine deaminase acting on RNA 2 (ADAR2), which is an enzyme that catalyzes the hydrolytic deamination of adenosine to inosine. As inosine is functionally equivalent to guanosine, such a construct can be optimized in genome engineering to induce desired base change at a specific nucleobase in the codon, useful for both research and clinical applications. It is worth noting that a hyperactive mutant of the wildtype ADAR2 (ADAR2<html><sub>DD</sub></html>), which has its glutamic acid at position 488 replaced by a glutamine (E488Q) is fused here, to allow looser stringency on the target sequence as well as to increase on-target efficiency. |
===Characterisation of Part=== | ===Characterisation of Part=== | ||
| − | In our project | + | In our project, we aimed to characterize the adenosine-to-inosine editing activities of this construct when targeting both the mRNA transcript of exogenous and the endogenous genes. Since it serves as a basis of comparison for the other construct, please refer to <html><a href="https://parts.igem.org/Part:BBa_K2818001"> this page </a></html> to view the methodology and results for its characterization. <br> |
| − | + | Also, since it is a published construct that has been well-characterized. | |
| − | + | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K2818002 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2818002 SequenceAndFeatures</partinfo> | ||
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Revision as of 16:52, 17 October 2018
Cas13b-NES-ADAR
dPspCas13b-ADAR2DD(E488Q) is a fusion protein that catalyzes the hydrolytic deamination of adenosine to form inosine in RNA molecules when used in conjunction with a guide RNA. Although there are preferred motifs, no Protospacer Adjacent Motif (PAM) is required. It is an optimized construct obtained developed by Zhang Feng's lab (Feng et. al., 2017) to mediate efficient adenosine to inosine on specific positions on mRNA target, which can be programmed by a specific guide RNA. This construct is submitted as it is currently not in the iGEM registry and it acts as an important basis for comparison when we characterize our new part this year (BBa_K2818001).
Usage and Biology
As mentioned above, the dPspCas13b is the catalytically inactive version of Type VI RNA-targeting CRISPR-associated protein 13b, an RNA-guided ribonuclease derived from Prevotella sep. P5-125 and it acts as the RNA-targeting scaffold to bind to specific RNA target sequence. Such a binding action is mediated by a single guide RNA, the sequence of which greatly affects the binding efficiency of dCas protein onto the target and ultimately the functionality of the fused protein.
The other domain fused to the dCas13b here is the Adenosine deaminase acting on RNA 2 (ADAR2), which is an enzyme that catalyzes the hydrolytic deamination of adenosine to inosine. As inosine is functionally equivalent to guanosine, such a construct can be optimized in genome engineering to induce desired base change at a specific nucleobase in the codon, useful for both research and clinical applications. It is worth noting that a hyperactive mutant of the wildtype ADAR2 (ADAR2DD), which has its glutamic acid at position 488 replaced by a glutamine (E488Q) is fused here, to allow looser stringency on the target sequence as well as to increase on-target efficiency.
Characterisation of Part
In our project, we aimed to characterize the adenosine-to-inosine editing activities of this construct when targeting both the mRNA transcript of exogenous and the endogenous genes. Since it serves as a basis of comparison for the other construct, please refer to this page to view the methodology and results for its characterization.
Also, since it is a published construct that has been well-characterized.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2884
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1787
Illegal BamHI site found at 839
Illegal BamHI site found at 3310
Illegal XhoI site found at 4011 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1520
Illegal AgeI site found at 2396 - 1000COMPATIBLE WITH RFC[1000]