Part:BBa_K2818002

Cas13b-NES-ADAR

dPspCas13b-ADAR2DD(E488Q) is a fusion protein that catalyzes the hydrolytic deamination of adenosine to form inosine in RNA molecules when used in conjunction with a guide RNA. Although there are preferred motifs, no Protospacer Adjacent Motif (PAM) is required. It is an optimized construct developed by Zhang Feng's lab (Cox et. al., 2017) to mediate efficient adenosine to inosine base change on specific positions of the mRNA target, which can be programmed by a specific guide RNA. This construct is termed as the REPAIR System (RNA Editing for Programmable A to I Replacement System) and it is currently not in the iGEM registry. Since it acts as an important basis for comparison when we characterize our new part this year (BBa_K2818001), we have submitted it with our own data for its characterization.

Usage and Biology

As mentioned above, the dPspCas13b is the catalytically inactive version of Type VI RNA-targeting CRISPR-associated protein 13b, an RNA-guided ribonuclease derived from Prevotella sep. P5-125 and it acts as the RNA-targeting scaffold to bind to specific RNA target sequence. Such a binding action is mediated by a single guide RNA, the sequence of which greatly affects the binding efficiency of dCas protein onto the target and ultimately the functionality of the fused protein.
The other domain fused to the dCas13b here is the Adenosine deaminase acting on RNA 2 (ADAR2), which is an enzyme that catalyzes the hydrolytic deamination of adenosine to inosine. As inosine is functionally equivalent to guanosine, such a construct can be optimized in genome engineering to induce desired base change at a specific nucleobase in the codon, useful for both research and clinical applications. It is worth noting that a hyperactive mutant of the wildtype ADAR2 (ADAR2_DD), which has its glutamic acid at position 488 replaced by a glutamine (E488Q) is fused here, to allow looser stringency on the target sequence as well as to increase on-target efficiency.

Characterisation of Part

In our project, we aimed to characterize the adenosine-to-inosine editing activities of this construct when targeting both the mRNA transcript of exogenous and the endogenous genes. Since it serves as a basis of comparison for the other construct, please refer to this page (BBa_K2818001) to view the methodology and results for its characterization. Also, since it is a published construct that has been well-characterized, the articles we referred to below also contains the characterization of this construct.

Reference

1. Kuttan, A., & Bass, B. L. (2012). Mechanistic insights into editing-site specificity of ADARs. Proceedings of the National Academy of Sciences, 109(48), E3295-E3304.
2. Cox, D. B., Gootenberg, J. S., Abudayyeh, O. O., Franklin, B., Kellner, M. J., Joung, J., & Zhang, F. (2017). RNA editing with CRISPR-Cas13. Science, 358(6366), 1019-1027

Contribution by Team NTU-Singapore iGEM 2019

Group: Team NTU-Singapore 2019
Author: Douglas Tay Jie Wen, Teo Seok Yee
Summary: For our bronze part characterisation, we added Sanger sequencing data to part BBa_K2818002 from Team NTU-Singapore iGEM 2018. Based on gRNA length optimisation (using luciferase assay) by our advisor, we found that decreasing the gRNA spacer length to 26 base pairs allows for 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.

Methodology

The experiment was conducted using gRNA targeting KRAS, PPIB, GAPDH or RAB7A mRNA (on-targets). Although KRAS and PPIB were characterized previously, the gRNA length 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 (except for off-targets) and Amplicon sequencing. To quantify editing for Sanger sequencing, we utilised the formula of % editing = Peak_G/(Peak_G + Peak_A).^[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 Results

Figure 1. Sanger chromatogram of KRAS. Editing = 7.5%.

Figure 2. Sanger chromatogram of PPIB. Editing = 64.9%.

Figure 3. Sanger chromatogram of GAPDH. Editing = 35.1%.

The boxed region in each chromatogram indicates the position of edit. As can be seen, the editing for KRAS is rather low at 7.5%, but the housekeeping genes PPIB and GAPDH had rather high edits of 64.9% and 35.1% respectively. The data for RAB7A was not shown due to noise in the chromatogram.

Amplicon Sequencing Results

Figure 4. Amplicon sequencing of various on-target genes. Adenosine positions are indicated for each gene. Heat map shows % of editing, the darker the red colouration, the higher the edit.

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. As seen, sites other than the intended target adenosine were also edited, showing that there is still much to be improved for dCas13b-ADAR2DD to increase its specificity. Data for PPIB was not shown as it had poor coverage of reads.

Figure 5. Amplicon sequencing of various off-target genes. Adenosine positions are indicated for each gene. Heat map shows total editing. The darker the red colouration, the higher the edit at that position.

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-ADAR2DD has high off-target activities, which could be improved upon by rational engineering.

Reference

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2884
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1787
  Illegal BamHI site found at 839
  Illegal BamHI site found at 3310
  Illegal XhoI site found at 4011
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1520
  Illegal AgeI site found at 2396
  
• 1000
  COMPATIBLE WITH RFC[1000]