eCRUIS captures RNA-protein interaction in vitro and in vivo
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Abstract
As an information bridge between DNA and protein, RNA regulates cellular processes and gene expression in a variety of ways. From synthesis to degradation, RNA is associated with a series of RNA-binding proteins. Therefore, it is very important to develop innovative methods to study the interaction between RNA and protein. Previously, we developed an RNA-centric method, called C RISPR-based R NA- U nited I nteracting S ystem (CRUIS), to capture RNA-protein interaction in cells. On this basis, here we develop an enhanced CRUIS (eCRUIS) by combining the power of dCas13d and the engineered promiscuous ligase TurboID. The new version allows us to label RNA-binding proteins on the target RNA within 30 minutes, which may be used in vivo. By introducing bait-assay with exogenous RNA, we confirm that eCRUIS can effectively label RNA-binding proteins on bait RNA in a short time. eCRUIS provides a wider range of in vitro and in vivo applications.
Article activity feed
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This study presents eCIRUS, and innovative method to capture RNA-protein interactions by proximity based biotin labelling of protein using dCas13d and joined ligase TurboID. While experiments (in vitro & in vivo) are carried out to show proof of concept, more analysis could be carried out on the data set acquired by deploying the method in vivo in transgenic Drosophila (what proteins were labelled? what was the target RNA used?), which could be highly insightful and could increase impact and interest in general.
This is an archived comment originally written by Januka Athukoralage
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labeling within 30 min
Were shorter or range of time points assessed, other than the 30 min timepoint after proximity labelling? While the authors conclude 30 min to be a short time, it is not immediately clear to me why this is, without having carried out a time course.
This is an archived comment originally written by Januka Athukoralage
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obviously activity
Consider revising to "obvious activity".
This is an archived comment originally written by Januka Athukoralage
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is considered to have extremely high RNA binding activity.
I am not sure what the authors mean by this, perhaps the authors should comment on the reported affinity of Cas13d for relevant targets.
This is an archived comment originally written by Januka Athukoralage
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dCas13a, which losses of RNA cutting activity
consider revising to "which lacks RNase activity".
This is an archived comment originally written by Januka Athukoralage
-
which the
consider revising to "consisting of"
This is an archived comment originally written by Januka Athukoralage
-
labeling within 30 min
Were shorter or range of time points assessed, other than the 30 min timepoint after proximity labelling? While the authors conclude 30 min to be a short time, it is not immediately clear to me why this is, without having carried out a time course.
-
obviously activity
Consider revising to "obvious activity".
-
is considered to have extremely high RNA binding activity
I am not sure what the authors mean by this, perhaps the authors should comment on the reported affinity of Cas13d for relevant targets.
-
dCas13a, which losses of RNA cutting activity,
consider revising to "which lacks RNase activity".
-
which the
consider revising to "consisting of"
-
This study presents eCIRUS, and innovative method to capture RNA-protein interactions by proximity based biotin labelling of protein using dCas13d and joined ligase TurboID. While experiments (in vitro & in vivo) are carried out to show proof of concept, more analysis could be carried out on the data set acquired by deploying the method in vivo in transgenic Drosophila (what proteins were labelled? what was the target RNA used?), which could be highly insightful and could increase impact and interest in general.
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