Part:BBa_K2818001

Cas13d-NLS-ADAR

Similar to part BBa_K2818002 (dPspCas13b-ADAR2_DD), dCas13d-ADAR2_DD(E488Q) is a fusion protein of ADAR2 adenosine deaminase 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 guide RNA sequence. It can be used to selectively edit adenosine to inosine in RNA molecules in the presence of the guide RNA. Nuclear Localization Signal was also added to facilitate localization of constructs in the nucleus and hence enhance RNA editing.

Team NTU-Singapore 2019 improved this part by lowering the off-target activity while retaining a relatively high on-target activity. Read more about it at BBa_K3250012.

Usage and Biology

As mentioned, this part is used to target RNA to edit a specific adenosine to inosine, when accompanied by a short guide RNA sequence. Similar to the dPspCas13b, the Cas13d domain here is the protein scaffold that targets and guides the same ADAR2 domain to the desired locations to perform hydrolytic deamination of adenosine to inosine. However, one great advantage of the Cas13d system over the Cas13b counterpart is its small size. With the average size of just 930 amino acids, it is the smallest Class 2 CRISPR effector (as of October 2018) 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 (Konermann, et. al., 2018). Hence, it is an interesting construct with great potential in useful real-life applications.

Improvement by AFCM-Egypt 2023 team

MCP-ADAR2 is an improved version of mutant form of ADAR2 enzyme (BBa_K2818001) that was designed by (iGEM18_NTU-Singapore) We conjugated the mutant form ADAR2dd to MS2 coat protein MCP, which specifically binds to short MS2 RNA hairpin 3D structure that replaces the promiscuous dsRNA-interacting domain of natural ADAR. Integrating MCP-ADAR and flanking the sensor UAG codon with two MS2 hairpins allows us to amplify the initial signal of our therapeutic cargo and replace the low background editing of natural ADAR by the targeted and efficient MCP-ADAR2 which ensures maximum activity of our therapeutic cargo within the target auto-reactive B-cells as shown in the following figure.

This figure illustrates the mechanism of MCP-ADAR recruitment toward the site of editing flanked by two MS2 RNA hairpin structures.

In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness. As displayed in the chart below, the mutation (Q173E) shows the highest epistatic fitness, while the lowest score was associated with the mutation (S51D).

Figure . An illustration of the effects of different mutations on the Epistatic Fitness of MCP ADAR.

Methodology of Characterization

We aimed to characterize the A-to-I editing activities on mRNA 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 reporter assay and direct targeting of 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 on mRNA, resulting in a nonsense mutation. As such, after transfection, A-to I editing activities on the mRNA transcript by the dCas-ADAR2_DD constructs 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_DD 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 are 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)

Figure 6. Editing rate of different RNA editors with different spacer lengths and different guide mismatch distances on endogenous KRAS mRNA. (n = 2)

Conclusion

Here we have demonstrated Type VI Cas13 proteins can mediate efficient A-to-I base editing on mRNA, for both exogenous 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 constructs as it has significantly smaller size and there is still the 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
4. Konermann, S., Lotfy, P., Brideau, N. J., Oki, J., Shokhirev, M. N., & Hsu, P. D. (2018). Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors. Cell, 173(3), 665-676.

Contribution by Team NTU-Singapore iGEM 2019

Group: Team NTU-Singapore 2019
Author: Ge Xiao Yu, Liew Kai Shin, Ng Zhi Jian
Summary: From our human practices discussions with professionals, we were reminded that RNA tends to form secondary and tertiary structures in cells. As our dCasRx-ADAR2DD editing is based on the formation of a duplex between the gRNA and the single-stranded target mRNA, they cautioned us that the presence of these structures in our target could inhibit gRNA binding, thereby preventing RNA editing. Thus, we sought to investigate whether RNA structure affects dCasRx-ADAR2DD RNA editing by comparing editing rates in open (linear) and structured (tend to form secondary/tertiary structures) RNA. icSHAPE reactivity profiles and scores reports on RNA structure at a transcriptome-wide level. Low and high icSHAPE scores are indicative of secondary and linear structures respectively. Here, we used icSHAPE reactivity scores and RNA expression levels to predict candidate RNAs that form linear or secondary structures. From icSHAPE data, we selected 20 open and 20 structured candidate RNAs with high expression levels to ensure sufficient reads for targeted editing by dCasRx-ADAR2DD.

Methodology

We designed gRNA to the 20 open and 20 structured RNAs. HEK293FT cells were transfected with BBa_K2818001 and a gRNA. After transfection, we performed PCR of the respective genes and barcoded for deep sequencing. We then looked at the number of reads and calculated editing% as G/(G+A).

Results

Figure 1. Editing on Open RNA.

Figure 2. Editing on Structured RNA.

Given that structured RNA play critical roles in cellular regulation and function, dCasRx-ADAR2DD-mediated RNA editing in structured RNA can have important implications for health and disease. Our results indicate that dCasRx-ADAR2DD has no clear preference for open and structured RNA. This means that dCasRx-ADAR2DD can target diverse RNA populations in the cell, making it useful for therapeutic and research applications. Another method that can more accurately predict RNA structures would be PARIS (psoralen analysis of RNA interactions and structures), which can resolve RNA structure at base pair level. In the future, PARIS information can be used to guide RNA structure prediction to further investigate the RNA editing activity of dCasRx-ADAR2DD for open and structured RNA.

References

1. Flynn R, Zhang Q, Spitale R, Lee B, Mumbach M, Chang H. Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE. Nature Protocols. 2016;11(2):273-290.
2. Chan D, Feng C, Spitale R. Measuring RNA structure transcriptome-wide with icSHAPE. Methods. 2017;120:85-90.
3. Lu Z, Zhang Q, Lee B, Flynn R, Smith M, Robinson J et al. RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure. Cell. 2016;165(5):1267-1279.

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]