Part:BBa_K4170029
crRNA targeting the miR-17-5P (extra loop design)
This part contains the sequences of the stem loop (repeat part) and the spacer (detection part) required for the formation of the CRISRPR/Cas13a complex and the hybridization with the mature miR-17-5P, respectively. The LbuCas13a protein adopts a bilobed structure which consists of a a-helical REC lobe and a NUC lobe. The crRNA stem loop sequence is anchored to the space between the NTD and Helical-1 domain forming extensive contacts between the crRNA and the LbuCas13a protein. This crRNA derives from the crRNA targeting the miR-17-5P (standard design-BBa_K4170027) but has a different design for the spacer sequence. The design of crRNA sequence is based on the introduction of an extra hairpin loop in the 3’ end of spacer sequence.
Sequence- crRNA 17-5p-hairpin design and in silico evaluation
The sequence is consisted of 2 main parts. The first part is called Repeat Part and it is a standard nucleotide sequence that binds to the Recognition Lobe of LbuCas13a and forms a stable complex with it. The second part is called spacer or detection part and it is responsible for the detection of the miR 17-5p. The Repeat part contains 31 nucleotides, and the spacer sequence is designed to contains 20 nucleotides that are complementary to the target miR sequence(Liu et al., 2017). This specific crRNA sequence have a different design for the spacer sequence. The design of crRNA sequence based on the introduction of an extra hairpin loop in the 3’ end of spacer sequence. This extra hairpin loop has been reported to reduce the off-target effects and undesired interactions of crRNA sequence with the target RNA sequence(Ke et al., 2021).The effect is described on the following Figure . The difference in Gibbs free energy between the off-target (undesired) interaction and the on-target interaction is increasing with the extra hairpin loop in the 3’ end of the spacer sequence. Bigger the difference between 2 energy states the more favorable will be the interaction with the minimum free energy and in this case the on-target interaction(Ke et al., 2021).The number of the nucleotides introduced to the 3’ end of the spacer sequence is 5, aiming to the formation of 5 base pairs and the number of nucleotides on the stem designed to be 4. This means that the 5 introduced nucleotides are reverse complementary to the nucleotides to position 43-47.
The first step of the in-silico evaluation is the evaluation of its thermodynamic properties. The software used for this task is ViennaRNA(Lorenz et al., 2011). The properties studied were : Minimum Free Energy, GC content of the sequence, Linearity of Detection Part, the Binding energy with the target miRNA and perfect matches.
The Minimum Free Energy is a measure of stability of the RNA and the negative it is the more stable will be. The
Vienna Software is calculating the energy based on the secondary optimal structure. The value of -12.8 kcal/mol
suggesting a relative stable structure.
GC content is ratio of the Guanine and Cytokine that are present in the sequence. The higher the ratio, the more
stable the sequence will be. This phenomenon is based on the different chemical groups that are present in every
nucleotide resulting stronger hydrogen bonds in comparison with other nucleotides and the value 0.48 is low since
values >0.5 are preferable in terms of stability.
Linearity Of Detection Part (LODP) shows the secondary structure of the detection part of the crRNA sequence.
Closer to 0 more linear will this part be. The linear structure of the detection part of crRNA is highly associated with the
proper function of the LbuCas13a protein. The value of -5.9 kcal/mol is the result of hydrogen bonds in the spacer
sequence that designed to formed in 3’end, but since the value is low the miRNA-crRNA sequence interaction is
energetically favorable.
Binding energy is the energy term of the difference between the Gibbs free energy of the complex RNA and the
free energy of crRNA and miRNA target. The binding energy is calculated using the secondary structure of the sequences.
Since the value -43.9 kcal/mol of the binding energy is nearly 3 times the MEF of the crRNA sequence then the stability
of complex is the preferable energetical state.
Perfect Matches is a measure of evaluation of the RNA complex that is formed by crRNA and miRNA target
sequences. The secondary structure of the complex is calculated through RNAduplex, and perfect matches is the ratio of
the nucleotides that are bind properly in detection part of the crRNA sequences. The ratio of 1.0 is showing that the
binding on the spacer sequence will be exactly as the suggested binding upon designing the sequence. The extra
introduced nucleotides did not affect the position of the interaction.
The predicted secondary structure of the crRNA sequence and the complex formed between the crRNA and
miRNA sequence are demonstrated on the next figure:
The next step for the in-silico evaluation is the molecular docking with the LbuCas13a protein. For this process, the utilized pdf file for LbuCas13a acquired from Protein Data Bank (ID 5XWY) and the pdf file for the crRNA 17-5p-hairpin sequence generated through the RNAComposer Sever. The docking algorithm was the HDOCK docking algorithm for the webserver(Zhang Di Yumeng Yan, 2017). The docking model selected was the best model that had the more negative value based on the scoring algorithm of HDOCK server(Wang et al., 2020).
The docking score is the energy score calculated through the scoring function of HDOCK algorithm.[4] The docking score is negatively related to the stability of the complex, meaning the more negative is the docking score more stable the complex receptor-ligand will be. The value of -349.98 kcal/mol shows that the complex formation is the energetical favorable state. The Ligand Root Mean Square Deviation is showing the difference between the positions of nucleotides of the ligand’s structure before and after docking. The ligand RMSD value of 47 Angstrom shows a major change in the conformation of the ligand which is acceptable since the ligand in this specific docking process is an RNA sequence of 55 nucleotides. The interaction site of the LbuCas13a based on the best model from HDOCK docking algorithm is the NUC Lobe Domain and the interaction site of the crRNA sequence is both the Repeat and the Detect Part of the crRNA 17-3p-hairpin. In comparison with the literature(Liu et al., 2017), the protein-RNA interactions are on the REC Lobe of the protein and the Repeat Part of the crRNA sequence suggesting that the docking model resulted to a different conformation of the 2 molecules.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Citations
1.Ke, Y. et al. (2021) ‘Hairpin-Spacer crRNA-Enhanced CRISPR/Cas13a System Promotes the Specificity of Single Nucleotide Polymorphism (SNP) Identification’, Advanced Science, 8(6), pp. 1–11. Available at: https://doi.org/10.1002/advs.202003611.
2.Liu, L. et al. (2017) ‘The Molecular Architecture for RNA-Guided RNA Cleavage by Cas13a’, Cell, 170(4), pp. 714-726.e10. Available at: https://doi.org/10.1016/j.cell.2017.06.050.
3.Lorenz, R. et al. (2011) ‘ViennaRNA Package 2.0’, Algorithms for Molecular Biology, 6(1), pp. 1–14. Available at: https://doi.org/10.1186/1748-7188-6-26.
4.Wang, L. et al. (2020) ‘Rapid design and development of CRISPR-Cas13a targeting SARS-CoV-2 spike protein’, Theranostics, 11(2), pp. 649–664. Available at: https://doi.org/10.7150/thno.51479.
5.Zhang Di Yumeng Yan (2017) ‘HDOCK : a web server for protein-protein and protein -DNA/RNA docking based on a hybrid strategy’.
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