Part:BBa_K2818001

Cas13d-NLS-ADAR

Similar to part BBa_K2818002 (dPspCas13b-ADAR2_DD), Cas13d-ADAR2DD(E488Q) is a similar fusion protein of ADAR2 adenosine deamination and a Type IV CRISPR-associated RNA-guided ribonucleases (RNase) 13d that is mutated to be catalytically inactive but retains the ability of binding to RNA target with a separate guide RNA sequence. It can be used to selectively edit adenosine to inosine in RNA molecules in the presence of a guide RNA. NLS was fused to improve localization in the nucleus and hence enhance RNA editing.

Usage and Biology

The possible applications and the working mechanism of Cas13d is similar to that the Cas13b, which is a protein scaffold to target and guide the ADAR2 domain to the desired location to perform hydrolytic deamination of adenosine to inosine. However, one great advantage of the Cas13d system is its small size. With the average size of just 930 amino acids, it is the smallest Class 2 CRISPR effector ever being characterized in mammalian cells. Despite its small size, the nuclease-dead variant derived from Ruminococcus flavefaciens XPD3002 (also known as CasRx) has demonstrated alternative splicing modulation in vivo with high efficiency and specificity. Hence, it is an interesting construct that can be potentially useful and promises great results.

Methodology for Characterization

We aimed to characterize both the A-to-I editing activities on transcripts of both the exogenous and endogenous genes, and compare it with the activities of the REPAIR system from literature. Two methods were used, namely a luciferase assay and direct targetting and sequencing of targeted endogenous mRNA.

Renilla luciferase Assay

In the luciferase reporter assay, the plasmid coding for a modified Renilla luciferase was constructed, where a guanosine is replaced by an adenosine at the codon of a key residue, resulting in a nonsense mutation. As such, after transfection, A-to I editing activities on the mRNA transcript by the dCas-ADAR2_DDconstructs will functionally restore the sequence and restores the luciferase protein back to the wildtype and allow for the quantification of editing activity by the Rluc luminescence. In our experiment, two parameters, namely spacer length and regions of coverage on the target were characterized in mediating A-to-I RNA editing.

Figure 1. Experimental design of luciferase assay

Endogenous mRNA Targetting

With the parameters obtained from the luciferase reporter assay, we further characterized the A-to-I editing activities of the dCas-ADAR2_DD constructs on endogenous mRNA. In such an experiment, plasmids coding for dPspCas13b-ADAR2_DD and dCas13d-ADAR2<tml>_DD</html> fusion proteins were transfected into HEK293FT cells, together with different guide RNAs targeting endogenous PPIB and KRAS mRNA transcripts. After 48 hours of transfection, the transcriptome of the cells was extracted and the target regions were amplified for Sanger sequencing. Fractions of the adenosine being called as guanosine and therefore being edited can then report for the on-target efficiency of A-to-I editing. Different guides were used to investigate the activities with different spacer lengths and guide mismatch locations.

Figure 2. Experimental design in endogenous mRNA targeting

Results of Characterization

Renilla luciferase Assay

In the luciferase experiment, we first evaluated the A-to-I editing activities of different RNA editors at different target positions on the Rluc mRNA. The aforementioned nonsense mutation of G to A was performed and tested at five tryptophan residues, at position 60, 104, 121, 153 and 219 respectively. Figure 3 below shows the luminescence levels after the restoration of Rluc sequence with different editors at different positions.

Figure 3. Editing rate of different RNA editors at different target positions (n = 2)

From the results, we can observe that except for REPAIRv2, other dCas-ADAR2DD constructs showed significant A-to-I editing activities on the target and showed different target preferences from dCas13b to dCas13d. For Cas13d, editing activities on Rluc W153X is particularly significant. Therefore, it is selected as the target position to investigate the effect of guide length and guide mismatch distance on the A-to-I editing activities.

Figure 4 shows the luminescence levels after the restoration of Rluc sequence by Cas13d using guides at different lengths and with different guide-target mismatch distance. The horizontal axis shows the mismatch distance and the number after items in the legend indicates different spacer length. Table 1 summarised all the observations made.

Figure 4. Editing rate of different RNA editors with different spacer lengths and different guide mismatch distances. (n = 2), where dash line shows the results for non-targeting control

From the results, we were then able to design homology-based guides with appropriate spacer length and guide mismatch distances and evaluate the performance of A-to-I editing on target mRNA.

Endogenous mRNA Targetting

In this part of the experiment, we used guides to target two different regions of the PPIB and KRAS mRNA. They were termed as guide 1 and 2 for PPIB and KRAS. Then, some of them will be given a suffix of X.Y, where X indicates the target length and Y indicates the guide mismatch distance. For example, KRAS-1-50.25 is the guide RNA targeting region 1 of KRAS with a spacer length of 50 base-pairs and a mismatch distance of 25 base-pairs. The following results were then obtained from Sanger sequencing. Editing rate is calculated as the area under the guanosine signal in the chromatograph over that of adenosine.

Figure 5. Editing rate of different RNA editors with different spacer lengths and different guide mismatch distances on endogenous PPIB mRNA. (n = 2)

Conclusion

Here we have demonstrated Type VI Cas13 proteins can mediate efficient A-to-I base editing on mRNA, for both exogeneous and endogenous transcripts. From both experiments we can conclude that while the optimized REPAIR enzyme showed higher A-to-I editing efficiency, unoptimized dCas13d-ADAR2DD constructs exhibited similar A-to-I editing activity level on mRNA on the PPIB loci. This shows great potential for the dCas13d-ADAR2DD construncts as it has significantly smaller size and there is still possibility for protein engineering and optimization.

Reference

1. Montiel-González, M. F., Vallecillo-Viejo, I. C., & Rosenthal, J. J. (2016). An efficient system for selectively altering genetic information within mRNAs. Nucleic acids research, 44(21), e157-e157.
2. Abudayyeh, O. O., Gootenberg, J. S., Essletzbichler, P., Han, S., Joung, J., Belanto, J. J., ... & Lander, E. S. (2017). RNA targeting with CRISPR–Cas13. Nature, 550(7675), 280.
3. 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

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2935
  Illegal BamHI site found at 2965
  Illegal XhoI site found at 2410
  Illegal XhoI site found at 3666
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 223
  
• 1000
  COMPATIBLE WITH RFC[1000]