Part:BBa_K3250012
dCasRx-ADAR2DD(D338)
Similar to part BBa_K2818001, dCasRx-ADAR2DD(D338) is a fusion protein of ADAR2 deaminase domain (with the existing E488Q hypermutation) 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
We have established 2 methods to measure and characterize the RNA editing activity of our constructs. First, we developed a luciferase reporter assay to assess exogenous RNA editing activity. Next, we performed Amplicon sequencing (Illumina) of endogenous target mRNA to assess endogenous RNA editing activity.
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.
We performed Amplicon sequencing (Illumina) of endogenous target mRNA (KRAS, PPIB, GAPDH and RAB7A) to assess on-target RNA editing. HEK293FT cells were co-transfected with plasmids encoding dCasRx-ADAR2DD and targeting gRNA.
In line with our aim of analysing for off-target editing, we also used a non-targeting (NT) gRNA (random sequence with no homology to the genome) to check for off-target editing in the XIAP, F11R and APOOL mRNA. ADAR substrates are normally dsRNA formed by self-complementarity, such as those containing Alu elements. These off-target genes were chosen as it has an Alu element and was reported to be a substrate of A-to-I editing in vivo. HEK293FT ADAR1 knockout cells were co-transfected with plasmids encoding dCasRx-ADAR2DD and NT gRNA.
Following a 48-hour incubation period, total RNA of the cells were extracted and converted to cDNA. Next, amplification of on-target editing and off-target editing sites were performed with primers targeting these sites to determine RNA editing efficiency and specificity of our dCasRx-ADAR2DD constructs. PCR products were then barcoded for Amplicon sequencing. To quantify editing, we used the formula of % editing = No. of reads in G/(No. of reads in G + No. of reads in A) x 100%.
Results
Luciferase
Figure 1. Luciferase data of RNA editing. Internal insertion of ADAR2 into CasRx.
Figure 1A shows the reversion of W60X to tryptophan, thus restoring the reading frame. Higher RLU indicates higher on-target edits. Figure 1B shows the off-target editing on *Rluc. Higher RLU indicates higher off-target edits. Based on our Luciferase reporter assay, we identified internal sites D338, G655 and E689 as optimal sites for ADAR2DD insertion as they have relatively high on-target RNA editing and lower off-target editing than dCasRx V1 (BBa_K2818001). Out of the 3 shortlisted internal sites, D338 appears to be the best site for ADAR2DD insertion as it has the highest on-target activity and lowest off-target activity.
Amplicon Sequencing
Figure 2. Amplicon sequencing reads for on-target genes (GAPDH, KRAS, PPIB, RAB7A).
Figure 3. Amplicon sequencing reads for off-target genes (F11R, APOOL, XIAP).
Figure 4. Scatter plot of average editing rate from amplicon sequencing data. Average editing rate calculated for all 4 on-target genes (GAPDH, KRAS, PPIB, RAB7A) and 3 off-target genes (F11R, APOOL, XIAP).
Data generated from amplicon sequencing was used to calculate average editing rates for 4 on-target genes (GAPDH, KRAS, PPIB, RAB7A) and 3 off-target genes (F11R, APOOL, XIAP). The average editing rates were plotted. dCasRx-ADAR2DD mutants that occur at the bottom right of the plot are ideal as they have high on-target and low off-target editing activity. Based on the plot (Figure 4), D338 seems to have very high on-target and relatively low off-target activity.
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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