Part:BBa_K3250019
dCasRx-ADAR2DD(D338+Q479L+K475D)
Similar to part BBa_K2818001, dCasRx-ADAR2DD(D338+Q479L+K475D) is a fusion protein of ADAR2 deaminase domain (with the existing E488Q hypermutation and additional Q479L+K475D mutations) and a Type VI CRISPR-associated RNA-guided ribonuclease, Cas13d. The Cas13d is mutated to be catalytically inactive but retains the ability of binding to the RNA target with a guide RNA (gRNA) sequence. As this Cas13d was derived from Ruminococcus flavefaciens XPD3002, we refer to this variant as CasRx (or dCasRx for the catalyically inactive ribonuclease). The ADAR2 is inserted within the coding region (loop) of dCasRx at D338. It can be used to selectively edit adenosine to inosine (A-to-I editing) in RNA molecules using a gRNA. A nuclear localization signal was also added to facilitate localisation of constructs in the nucleus for editing of RNA transcripts.
Usage and Biology
dCas13d or dCasRx is the catalytically inactive version of Type VI RNA-targeting CRISPR-associated protein 13d, an RNA-guided ribonuclease derived from Ruminococcus flavefaciens. The main functional purpose of this part is to perform Adenosine to Inosine residue editing on targeted and specific Adenosine residues. Akin to the registered dPspCas13b, the Cas13d domain in this part refers to the protein scaffold that is responsible for specific binding to a target sequence through a gRNA complex and hence guiding the ADAR2 domain to the desired location(double stranded RNA region) to perform hydrolytic deamination. Due to its relatively small size as compared to Cas13b, a Cas13d system may be easier to modify, package and deliver, making it more suitable for clinical applications. For this part, a hyperactive mutant of ADAR2(E488Q) with its glutamic acid at amino acid position 488 replaced by a glutamine is fused here. This is to allow for greater flexibility of the target sequence and to achieve higher on-target efficiency.
Methodology
In our luciferase reporter assay, a plasmid (*Rluc) encodes a Renilla luciferase gene in which a guanosine (G) is replaced by an adenosine (A). This disrupts the luciferase gene, causing it to be nonfunctional (W60X mutation; tryptophan to a stop codon at position 60). When the *Rluc plasmid is co-transfected along with our dCasRx-ADAR2DD constructs and a gRNA targeting *Rluc, ADAR2DD converts A>I(G), which restores the luciferase gene and generates luminescence (X60W; stop codon to tryptophan). This allows for RNA editing activity to be quantified.
Results
Figure 1. Luciferase data for 5 ADAR2 mutants that were inserted into the site D388 on dCasRx.
As D338 had lower off-target and relatively high on-target activity compared to the control (dCasV1), we sought to further improve it through incorporating mutations into the ADAR2 deaminase domain. However, the luciferase data shows that D338 is still better on its own.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 4186
Illegal XhoI site found at 1776
Illegal XhoI site found at 3661 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 223
- 1000COMPATIBLE WITH RFC[1000]
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