Part:BBa_K3250002
dCasRx-ADAR2DD (K475D)
Similar to part BBa_K2818001, dCasRx-ADAR2DD(K475D) is a fusion protein of ADAR2 deaminase domain (with the existing E488Q hypermutation and an additional K475D mutation) 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). 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 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
Figure 1. Amplicon sequencing reads for on-target genes (GAPDH, KRAS, PPIB, RAB7A).
Figure 2. Amplicon sequencing reads for off-target genes (F11R, APOOL, XIAP).
Figure 3. 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 3), K475D seems to have relatively high on-target and low off-target activity.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2935
Illegal XhoI site found at 2410
Illegal XhoI site found at 3693 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 223
- 1000COMPATIBLE WITH RFC[1000]
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