Part:BBa_K4611006

crRNA 2 targeting the miR-21-5p under T7 promoter

crRNA is used to combine with the LwaCas13a protein in the CRISPR system, recognizing miR-21-5p to activate LwaCas13a.The crRNA full length spacer region is the reverse complementary sequence of miR-21-5p. This part is DNA template of crRNA 2. It is designed with sequence of T7 promoter. Transcribing it with T7 RNA polymerase will obtain crRNA2.

Model

Evaluation of crRNA

To anticipate the stability of crRNA, we evaluated crRNA in three ways: 1) Minimum Free Energy; 2) GC content; 3) MFE heterodimers

crRNA	MFE (kcal/mol)	GC content	MFE heterodimer (kcal/mol)
crRNA1	-9.8	0.39	-41.6
crRNA2	-9.7	0.41	-40.9
crRNA3	-9.6	0.41	-39.8
crRNA full length	-9.6	0.40	-42.5

Table 1 Simulation parameters for crRNA

Figure 1 Secondary structure of crRNA. (A) crRNA1; (B) crRNA2; (C) crRNA3; (D) crRNA full length(Green: Stems, Yellow: Interior Loops, Blue: Hairpin loops, Orange: 5' and 3' unpaired region)

The stability of crRNA can be described using two factors: the Minimum Free Energy (MFE) and the GC content. A smaller MFE value and a larger GC content value indicate a more stable crRNA. In addition, the MFE Heterodimer is used to describe the stability of the binary complex formed between crRNA and miRNA. A smaller MFE Heterodimer value indicates a more stable binary complex formed by the combination of crRNA and miRNA. The stability of both the crRNA structure and binary complex structure are essential for the proper functioning of the CRISPR/Cas13a system. Our findings indicate that the stability of crRNA full length is better, as is the stability of the complex formed by crRNA and miRNA.

Molecular Docking

Molecular docking is a method of predicting receptor-ligand interactions. Based on the properties of receptor and ligand molecules, various possible conformations are docked and scored based on energy and binding conditions.

We obtained the PDB file of LwaCas13a protein by homology modeling. Then, we used the 3d RNA server to obtain the tertiary structure and PDB file of crRNA from its previously obtained secondary structure.

Figure 2 Homologous modeling of proteins & Preparation of docking files (A) The crystal structure of LbuCas13a; (B) A homology modeling structure of LwaCas13a; (C) A superimposed image of LbuCas13a and LwaCas13a; (D) Structural domain annotation of LwaCas13a.

Figure 3 Predicted structure of crRNA with 3dRNA. (A) crRNA1; (B) crRNA2; (C) crRNA3; (D) crRNA full length.

After obtaining the PDB files for LwaCas13a and crRNA required for molecular docking, we performed rigid docking of the LwaCas13a protein and crRNA using HDOCK server. The server provided scoring and RMSD for each docking.

Figure 4 Docking scores for crRNA and LwaCas13a proteins calculated by HDOCK

Figure 5 RMSD for crRNA and LwaCas13a proteins calculated by HDOCK

The Docking Score is a widely-used evaluation metric in HDOCK for assessing molecular docking. The metric measures the capacity of a docking simulation to accurately predict the binding between two molecules. A lower Docking Score indicates a better docking performance. RMSD (Root Mean Square Deviation) is a parameter that quantifies the deviation of atoms from their expected positions during molecular docking. A lower RMSD value corresponds to a more stable docking. By integrating these two parameters, it was observed that crRNA full length exhibited superior performance in molecular docking.

Molecular Dynamic Simulation

To assess the stability of the CRISPR/Cas system, we conducted molecular dynamics simulations utilizing the GROMACS software. We set various parameters to ensure the simulation conditions closely resembled the actual scenario.

After completing the simulation, GROMACS tools were used to analyze the trajectory and calculate the root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent accessible area (SASA), and hydrogen bonds (Hbonds)

The Root Mean Square Deviation (RMSD) is an important measure of the positional variance of the atoms in a molecule with respect to a reference position. A small and stable RMSD value indicates a high degree of stability in the molecule.

The Root Mean Square Fluctuation (RMSF) is a measure of the average position of atoms in a molecule over time, which indicates the degree of flexibility of the molecule being analyzed during the simulation. Generally, a larger RMSF value implies a higher degree of flexibility of a particular region of the molecule and indicates its relative distance from the active position.

Hydrogen bonds are capable of forming both within individual molecules and between multiple molecules. The stability of a molecule can be determined by the number of hydrogen bonds present. In the context of molecular dynamics simulations, a consistent count of hydrogen bonds is a strong indication of the molecule's stability.

The solvent accessible surface area (SASA) is a parameter that is closely associated with the solvation free energy of a molecule. Additionally, it can serve as a measure of the stability of macromolecules when placed in different solvents.

The calculation of four parameters for the crRNA was the first step of our analysis

Figure 6 Molecular dynamics simulation parameters of crRNA full length. ( A. RMSD of crRNA full length, B. RMSF of crRNA full length, C. SASA of crRNA full length, D. Hbonds of crRNA full length )

The findings demonstrate that the parameters of crRNA are characterized by good stability, indicating that the three-dimensional structure of crRNA is relatively stable.

The principle of this method is as follows:

Figure 7: Schematic presentation of cloning strategy of Pveg plasmid with full length crRNA sequences

PCR amplification:

PCR products encoding the part of crRNA loop region were obtained by using loop primers and using Pveg plasmid as template. This set of primers amplifies the portion of the plasmid containing the BioBrick prefix and CmR.

PCR products encoding the part of crRNA guide sequence were obtained by using spacer primers and using Pveg plasmid as template. This set of primers amplifies the portion of the plasmid containing the BioBrick suffix and Ori.

Golden Gate Assembly:

The hanging ends of all primers were designed with BsaI endonuclease digestion sites, so the PCR products obtained also had BsaI digestion sites at both ends. After BsaI digestion the PCR product will have designed sticky ends at both ends. The PCR product will be ligated by T4 ligase to form a new plasmid.

Figure 8 Two sets of PCR products can be assembled by Golden Gate to obtain a complete plasmid containing the coding sequence of crRNA. After amplification and linearization, crRNA can be obtained by in vitro transcription using T7 RNA polymerase.

Experiment

First of all, we designed two sets of primers: Loop and Spacer. Primers loop contained the overhanging part and the binding part. The overhanging part was used to produce the DNA template of the crRNA loop sequence, and the binding part was combined with sequence of the plasmid; Primer Spacer also contained overhanging part and the binding part, and the overhanging part was used to produce the DNA template of the crRNA guide sequence, and the binding part combined with sequence of the plasmid. All PCR steps followed the instructions of the PrimeSTAR® HS DNA Polymerase(Takara) kit, using the 3step method.

PCR:

• Using primer loop F and loop R, the pveg plasmid was used as template for PCR amplification.
• Using primer spacer F and sapcer R, the pveg plasmid was used as template for PCR amplification.

Gel electrophoresis verification was carried out after PCR (1% gel electrophoresis). After verifying the success of PCR, the gel blocks containing PCR products were cut off and the product was recovered from gel.

Figure 9 Gel electrophoresis after PCR of two set primers. Lane1: DNA marker. Lane2: Guide sequence of crRNA1. Lane3: Guide sequence of crRNA2. Lane4: Guide sequence of crRNA3. Lane5: Guide sequence of crRNA full length. Lane6: Loop region.

Golden Gate Assembly:

1. Golden Gate Assembly: the recovered product was ready for golden gate assembly system, and the experiment was carried out according to the golden gate assembly experimental scheme provided by SEVA 3.1: enabling interoperability of DNA assembly among the SEVA, BioBricks and Type IIS restriction enzyme standards.
2. Plasmid transformation: The plasmid obtained after assembly was transformed into the bacterial chassis(Trans1 T1 E.coil).
3. Colony PCR: LB solid medium with chloramphenicol was used for colony screening, and then colony PCR was performed to verify the success of Golden Gate assembly and plasmid transformation.

Figure 10 （A）PCR verifies that the plasmid has been successfully assembled.（B） Colony gel electrophoresis after PCR. Lane1: DNA Marker. Lane2-4: crRNA full length. Lane5-7: crRNA1. Lane8-10: crRNA2. Lane11-13: crRNA3

In vitro transcription:

1. Extraction and purification of DNA: the transformed colonies were cultured overnight. After culture, the colonies were cleaved to get DNA which would be purified to obtain the assembled plasmid for in vitro transcription
2. Plasmid linearization: the obtained plasmid was digested with SapI to linearize the plasmid to get DNA template.
3. In vitro transcription: this DNA template was transcribed in vitro using T7 RNA polymerase.

Figure 11: Gel electrophoresis plot of in vitro transcription of crRNA. Lane1: crRNA full length. Lane2: crRNA1.Lane3: crRNA2. Lane4: crRNA3.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]