Contribution

Overview

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As part of the DIAS project, we have developed several experimental methods and many new genetic parts in the belief that they would contribute to future iGEM teams who intend to utilize the CRISPR/Cas13a system. Whilst these contributions could allow the facile design and experimental implementation of all the biological “components” required to build a functional CRISPR/Cas13a system for disease detection, they could be also easily utilized for different applications and challenges. This page summarizes all the significant contributions provided by our team during our synthetic biology journey. Our team contributions to the synthetic biology community are briefly listed below:

• Design and documentation of new biological parts.
• New documentation to an existing part.
• A methodology for the in silico design and production of any crRNA based on the desired target miRNA in an easy, efficient and low-cost procedure. This methodology allows researchers to devote less time to design and clone the crRNA sequences, accelerating the time required to obtain useful results.

Design and documentation of new biological parts.

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During the developmental process of the DIAS diagnostic platform, we have developed several new genetic parts useful for CRISPR/Cas13a system implementation. This part collection contains the necessary codon optimized coding sequences for LbuCas13a production and purification in E.coli bacterial cells allowing researchers to recover the protein either from Inclusion bodies (IBs) or the cytoplasmic soluble fraction. In addition, this collection contains several crRNA sequences which were in silico designed to confer different binding properties to the miR-17-3p and miR-17-5p. Below, we provide briefly some information regarding the design and documentation of some parts that we efficiently produced and characterized. However, the overall information regarding the in silico design, the proposed cloning strategy and the characterization of these parts are provided on the corresponding part's registry page.

BBa_K4170016 - SUMO-LbuCas13a coding device under T7 promoter

This composite part contains the SUMO-LbuCas13a coding device under the transcriptional control of the T7 promoter. This part contains all the basic genetic parts required for the bacterial expression of codon-optimized LbuCas13a protein in fusion with small ubiquitin-like modifier (SUMO) protein which favors the increased expression and solubility of the recombinant LbuCas13a protein. The registry page of this part contains useful information that can be used by future iGEM teams which intend to use the CRISPR/LbuCas13a system as a part of their project. Specifically, we provide details regarding the biological function and usage of this part and the thorough cloning methodology that we followed to assemble this device into the pSB1C3 plasmid backbone. All cloning steps are accompanied by the corresponding results of the agarose gel electrophoresis.

In addition, a detailed documentation regarding the LbuCas13a protein expression and purification are depicted and can aid future synthetic biology researchers to succeed in efficient production and purification of this protein. For example, during our experiments we concluded that the 4 hour induction with 1mM IPTG at 37०C cannot facilitate the expressed protein localization in the soluble cytoplasmic fraction, on the contrary the insoluble fraction (IBs) was observed to contain the protein of interest. In addition, we investigated the IPTG induction time and the concentration of the added IPTG which is required to succeed effective LbuCas13a localization at the soluble cytoplasmic fraction. The results of our experiments demonstrated that after the 6 hour time interval the protein starts to localize at the soluble cytoplasmic fraction (Figure 1 lane 7) and the 1mM IPTG provides the highest yield of the expressed protein (Figure 2 Lane 2).

Furthermore, on the corresponding registry page and on the results page of our Wiki, we thoroughly describe the methods that we followed to construct a functional CRISPR/Cas13a detection system. Documentations are provided regarding the experimental conditions that we followed for the assembly of the molecular device such as the reaction buffer required for the LbuCas13a enzyme, the RNA reporter concentration, the quantification standard curve construction, etc. The results of the in silico Michaelis Menten analysis of our system and the experimental data from the conducted experimental assays could contribute to the overall scientific community.

BBa_K4170019 - crRNA targeting the miR-17-3P (standard design) under T7 promoter

This part contains the genetic device required for the in vitro transcription of the crRNA (standard design) required for the CRISPR/LbuCas13a-based detection of the miR-17-3P. A detailed cloning methodology concerning the construction of the crRNA coding device under T7 promoter (BBa_K4170019) for in vitro transcription is described on the parts registry and on the crPrep crRNA preparation kit. Information regarding the in silico design, the evaluation of the crRNA thermodynamic properties and the molecular docking studies of the LbuCas13a/crRNA complex are provided on the registry.

New documentation to an existing part.

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During the optimization process for the efficient LbuCas13a protein production and purification, we added extra information to the existing registry part Cas13a Lbu (BBa_K2926001) designed by iGEM19_Bielefeld-CeBiTec team. This part codes for Cas13a Lbu derived from Leptotrichia buccalis and according to the part's documentation in the registry the EcoRI and PstI site have been removed to succeed RFC [10] compatibility. This part was initially used by Bielefeld-CeBiTec team for assays in S.cerevisiae. This part was utilized by our team to conduct comparative experiments with the part BBa_K4170016 - SUMO-LbuCas13a coding device under T7 promoter which we constructed as an optimization effort to enhance maximum LbuCas13a expression efficiency in the cytoplasmic soluble fraction of the E.coli. We incorporated this part (BBa_K2926001) into the pSB1C3 plasmid backbone under transcriptional control of the T7 promoter, transformed the plasmid into BL21 (DE3) E.coli strain and induced the expression of the recombinant protein observing the protein solubility. The Cas13a Lbu coding device inserted into the pSB1C3 backbone generated a new composite part which is deposited at the IGEM Registry (BBa_K4170056)

Methodology for the in silico design and experimental production of any desired crRNA.

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The first step for CRISPR/Cas13a practical implementation is the standardization of the experimental conditions such as the suitable LbuCas13, reporter RNA and crRNA concentrations in the reaction, the appropriate handling procedures and the determination of the appropriate fluorescence acquisition time point since reaction initiation. This preliminary step could enhance the detection assay's performance and reliability.

After the above is achieved, then different crRNA sequences can be designed to achieve the detection of different target miRNA expanding the applications of the detection system. Examining thoroughly the crRNA sequence, one can easily understand that the only nucleotides that need to be modified are those that correspond to the protospacer, keeping constant the nucleotide sequence which forms the constant crRNA stem loop region that binds to Cas13a enzyme (Watanabe et al., 2019).

Seeking to simplify the in silico design and experimental process for the production of a functional miRNA-targeted crRNA, we introduce the crPrep crRNA preparation kit. The provided guidelines cover the entire crRNA developmental process from in silico design to in vitro transcription and allow future iGEM teams to efficiently and easily produce the desired crRNA even if they do not have significant expertise in designing genetic sequences for cloning applications.

crPrep crRNA preparation kit: method and guidelines

The basic principle of the method is the PCR amplification of any pSB#X# biobrick plasmid utilizing two sets of standardized primers. These primers have additional overhang sequences which correspond to the sequences of the crRNA spacer and loop respectively flanked by the BsaI recognition sequences on their 5' edge. After PCR amplification of the pSB1C3 plasmid, the first primer set incorporates the loop region of the crRNA in the amplified DNA product, while the second pair incorporates the desired crRNA spacer region in the corresponding PCR product. In addition, each primer set integrates suitable BsaI recognition sites on the edges of the amplified PCR products generating sticky ends which allow for the efficient assembly of the two amplified DNA sequences utilizing Golden Gate assembly.

• The loop FWD standard primer hybridizes with the pSB1C3 DNA region which corresponds to the sequence presented between the origin of replication (ori) and lambda t0 terminator of the pSB1C3 backbone.
• The loop RVS standard primer hybridizes with the pSB1C3 DNA region which corresponds to the biobrick prefix sequence of the pSB1C3 plasmid. This primer has an additional overhang sequence which corresponds to the T7 promoter along with the crRNA stem loop region.
• The spacer FWD interchangeable primer hybridizes with the biobrick suffix sequence of the pSB1C3. This primer has an additional overhang which corresponds to the desired crRNA spacer region. This region needs to be modified depending on the desired target miRNA of the CRISPR/Cas13a system.
• The spacer RVS standard primer hybridizes with the 3' edge of the pSB1C3 origin of replication.

As described above, 3 of the 4 required primers show conserved and identical nucleotide sequences regardless of the target miRNA. Thus, the only primer that needs to be modified according to the target miRNA is the spacer FWD interchangeable primer. The crPrep contains the reagents that are depicted on Figure 3.

In silico design of the spacer FWD interchangeable primer

As described above, the only primer which needs modifications depending on the desired target miRNA sequence is the spacer FWD interchangeable primer. The sequence noted with red bold color (Figure 4) should be replaced by the DNA sequence which is complementary to the miRNA target, however with 3' to 5' direction i.e upside down (Figure 5).

Guidelines on cloning strategy.

To efficiently construct the final plasmid with the desired crRNA insert under the transcriptional control of the T7 promoter (Moll et al., 2004), the following steps should be followed:

Step 1: PCR amplification

• PCR amplification with loop RVS standard and loop FWD standard primers using the PSB1C3 plasmid as a template. These primers amplify a specific region of the pSB1C3 plasmid sequence (lambda to terminator, CmR-chloramphenicol acetyltransferase, prefix) and introduce the loop sequence of the crRNA to the amplified PCR product. This PCR produces the loop part ready for Golden Gate assembly. The loop part is ready to use for any crRNA constructed with this method since the stem-loop sequence is universal for all crRNAs.
• PCR amplification with spacer FWD interchangeable and spacer RVS standard primers using the PSB1C3 plasmid as a template. These primers amplify a specific region of the pSB1C3 plasmid sequence (ori-origin of replication, his operon terminator, suffix) and introduce the spacer sequence of the crRNA to the amplified product. This PCR produces the spacer part ready for Golden Gate assembly.

Step 2: Golden Gate assembly

• Golden Gate-based SevaBrick assembly (Damalas et al., 2020) of the PCR amplified loop part and spacer part for the efficient construction of the crRNA coding sequence under the transcriptional control of the T7 promoter in the pSB1C3 plasmid.

Step 3: Bacterial transformation and plasmid DNA extraction

• The assembly reaction is transformed into chemically competent E.coli DH5a cells using heat shock transformation.
• Isolation of the crRNA-pSB1c3 plasmid using any commercial plasmid DNA isolation and purification kit.

Guidelines for in vitro Transcription.

Step 1: Linearization and purification

• Plasmid linearization utilizing the SapI restriction enzyme. To produce the crRNA with the defined length, the plasmid DNA should be linearized downstream of the inserted crRNA sequence.
• Linearized DNA template band purification utilizing any commercial DNA Gel Extraction Kit
• DNA template purification by phenol/chloroform extraction

Step 2: In vitro transcription

• The purified linear DNA template serves as a template for in vitro transcription utilizing T7 RNA polymerase. Any commercially in vitro transcription kit can be used to synthesize sufficient amounts of the desired crRNA.
• crRNA purification with any available RNA purification kit which allows for the efficient purification of small length RNAs.