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 IV 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-iGEM 2019

For our bronze part characterisation, we added Sanger sequencing data to part BBa_K2818002 from Team NTU-iGEM 2018. This part is a dCas13b-ADAR2DD fusion, which allows dCas13b-directed RNA editing using a target-specific gRNA. The effector domain for RNA editing is ADAR2DD, which catalyses the deamination of adenosine to inosine (A-to-I editing). Inosine is read as a guanosine, which essentially 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 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.

The experiment was conducted using gRNA targeting KRAS, PPIB and GAPDH mRNA. Although KRAS and PPIB were characterized previously, the gRNA length they used were greater than 26 base pairs. To determine whether shortening the gRNA length would also result in higher editing in endogenous genes, we tested the 26 base pair gRNA on KRAS and PPIB for comparison.

In this experiment, we transfected HEK293FT (ADAR1 knockout) cells with plasmids encoding dCas13b-ADAR2DD (BBa_K2818002) and the respective gRNA. Cells were lysed and harvested after 48 hours of transfection, and total RNA was extracted. The target regions (KRAS, PPIB and GAPDH) were then amplified for Sanger sequencing. To quantify editing, we utilised the formula of editing = PeakG/(PeakG + PeakA).

Results

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]