Difference between revisions of "Part:BBa K2818001"

Revision as of 17:24, 17 October 2018

Cas13d-NLS-ADAR

Similar to part BBa_K2818002 (dPspCas13b-ADAR2_DD), Cas13d-ADAR2DD(E488Q) is a similar fusion protein of ADAR2 adenosine deamination and a Type IV CRISPR-associated RNA-guided ribonucleases (RNase) 13d that is mutated to be catalytically inactive but retains the ability of binding to RNA target with a separate guide RNA sequence. It can be used to selectively edit adenosine to inosine in RNA molecules in the presence of a guide RNA. NLS was fused to improve localization in the nucleus and hence enhance RNA editing.

Usage and Biology

The possible applications and the working mechanism of Cas13d is similar to that the Cas13b, which is a protein scaffold to target and guide the ADAR2 domain to the desired location to perform hydrolytic deamination of adenosine to inosine. However, one great advantage of the Cas13d system is its small size. With the average size of just 930 amino acids, it is the smallest Class 2 CRISPR effector ever being characterized in mammalian cells. Despite its small size, the nuclease-dead variant derived from Ruminococcus flavefaciens XPD3002 (also known as CasRx) has demonstrated alternative splicing modulation in vivo with high efficiency and specificity. Hence, it is an interesting construct that can be potentially useful and promises great results.

Methodology for Characterisation

We aimed to characterize both the A-to-I editing activities on transcripts of both the exogenous and endogenous genes, and compare it with the activities of the REPAIR system from literature. Two methods were used, namely a luciferase assay and direct targetting and sequencing of targeted endogenous mRNA.

Methodology for Characterisation

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 2935
Illegal BamHI site found at 2965
Illegal XhoI site found at 2410
Illegal XhoI site found at 3666
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 223
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2818001 short</partinfo>
-Similar to part<html><a href="https://parts.igem.org/Part:BBa_K2818001"> BBa_K2818002 (dPspCas13b-ADAR2<sub>DD</sub>)</a></html>, Cas13d-ADAR2DD(E488Q) is a similar fusion protein of a Type IV that can be used to selectively edit adenosine to inosine in RNA molecules when used in conjunction with a guide RNA. NLS was fused to improve localisation to nucleus and hence RNA editing.
+Similar to part<html><a href="https://parts.igem.org/Part:BBa_K2818001"> BBa_K2818002 (dPspCas13b-ADAR2<sub>DD</sub>)</a></html>, Cas13d-ADAR2DD(E488Q) is a similar fusion protein of ADAR2 adenosine deamination and a Type IV CRISPR-associated RNA-guided ribonucleases (RNase) 13d that is mutated to be catalytically inactive but retains the ability of binding to RNA target with a separate guide RNA sequence. It can be used to selectively edit adenosine to inosine in RNA molecules in the presence of a guide RNA. NLS was fused to improve localization in the nucleus and hence enhance RNA editing.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+The possible applications and the working mechanism of Cas13d is similar to that the Cas13b, which is a protein scaffold to target and guide the ADAR2 domain to the desired location to perform hydrolytic deamination of adenosine to inosine. However, one great advantage of the Cas13d system is its small size. With the average size of just 930 amino acids, it is the smallest Class 2 CRISPR effector ever being characterized in mammalian cells. Despite its small size, the nuclease-dead variant derived from Ruminococcus flavefaciens XPD3002 (also known as CasRx)  has demonstrated alternative splicing modulation in vivo with high efficiency and specificity. Hence, it is an interesting construct that can be potentially useful and promises great results.
+===Methodology for Characterisation===
+We aimed to characterize both the A-to-I editing activities on transcripts of both the exogenous and endogenous genes, and compare it with the activities of the REPAIR system from literature. Two methods were used, namely a luciferase assay and direct targetting and sequencing of targeted endogenous mRNA.
+====Methodology for Characterisation====
 <!-- -->