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Characterisation

Bronze Medal Criterion #5

For our bronze part characterisation, we added Sanger sequencing data to part BBa_K2818002 from Team NTU-Singapore iGEM 2018. This part is a dCas13b-ADAR2_DD fusion, which allows dCas13b-directed RNA editing using a target-specific gRNA. The effector domain for RNA editing is ADAR2_DD, which catalyses deamination of adenosine to inosine (A-to-I editing). Inosine is read as a guanosine during translation, which results in an A>G base change in the RNA molecule.

Methodology

Based on gRNA length optimisation (using luciferase assay) by our advisor Yuan Ming, we found that decreasing the gRNA spacer length to 26 base pairs allows for a higher percentage of editing. The gRNA is still designed with an internal cytosine mismatch to the adenosine on the target site. This would cause ADAR2 to preferentially edit the adenosine at this position.

The experiment was conducted using gRNA targeting KRAS, PPIB, GAPDH or RAB7A mRNA (on-targets). Although KRAS and PPIB were characterized previously, the gRNA spacer they used were greater than 26 base pairs. Thus, we aim to include additional data using the new truncated 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.^[1,2] 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.

In this experiment, we transfected HEK293FT cells with plasmids encoding dCas13b-ADAR2_DD (BBa_K2818002) and the respective 26 base pairs gRNA (on-target or NT gRNA). Cells were lysed and harvested after 48 hours of transfection, and total RNA was extracted. The target regions were then amplified for Sanger and Amplicon sequencing. To quantify editing for Sanger sequencing, we utilised the formula of % editing = Peak_G/(Peak_G + Peak_A) x 100%.^[3-5] For Amplicon sequencing, we found the coverage of each adenosine and guanosine at the target sites, and calculated based on G/(G+A) x 100%.

Results

Sanger Sequencing Results

KRAS

Editing: 7.5%

PPIB

Editing: 64.9%

GAPDH

Editing: 35.1%

The boxed region in each chromatogram indicates the position of edit. From the above chromatograms, the editing for KRAS is fairly low at 7.5%, but the housekeeping genes PPIB and GAPDH had relatively high edits of 64.9% and 35.1% respectively. The data for RAB7A is not shown due to noise in the chromatogram.

Amplicon Sequencing Results

Amplicon sequencing of on-target genes.

The position of edits for GAPDH, KRAS and RAB7A are 83, 124 and 68, respectively. GAPDH had an editing rate of 30%, KRAS 25%, and RAB7A 30%. As amplicon sequencing is more sensitive than Sanger, we were able to identify cis off-targets. From the above heat maps, sites other than the intended target adenosine were also edited, showing that there is still much to be improved for dCas13b-ADAR2_DD to increase its specificity. Data for PPIB is not shown as it had poor coverage of reads.

Amplicon sequencing of off-target genes.

We transfected HEK293FT ADAR1 knockout cells with NT gRNA to investigate off-target editing at F11R, APOOL and XIAP. The heat map shows that various adenosines on these genes were edited compared to the untransfected control. This further indicates that dCas13b-ADAR2_DD has high off-target activities, which could be improved upon by rational engineering.

References

1. Franzén O, Ermel R, Sukhavasi K, Jain R, Jain A, Betsholtz C et al. Global analysis of A-to-I RNA editing reveals association with common disease variants. PeerJ. 2018;6:e4466.
2. Nishikura K. A-to-I editing of coding and non-coding RNAs by ADARs. Nature Reviews Molecular Cell Biology. 2015;17(2):83-96.
3. Eggington J, Greene T, Bass B. Predicting sites of ADAR editing in double-stranded RNA. Nature Communications. 2011;2(1).
4. Fritzell K, Xu L, Otrocka M, Andréasson C, Öhman M. Sensitive ADAR editing reporter in cancer cells enables high-throughput screening of small molecule libraries. Nucleic Acids Research. 2018;47(4):e22-e22.
5. Katrekar D, Chen G, Meluzzi D, Ganesh A, Worlikar A, Shih Y et al. In vivo RNA editing of point mutations via RNA-guided adenosine deaminases. Nature Methods. 2019;16(3):239-242.