Best Part Collection

Overview

Standardization, modularity and abstraction are some of the fundamental engineering principles applied by synthetic biology facilitating rapid prototyping and easy interchange of engineering designs between researchers. The interchangeable genetic elements designed in a modular way resemble toy building blocks that can be combined and modified in a fast and efficient way (Garner, 2021). The main elements that constitute the DIAS detection platform are the LbuCas13a enzyme, the crRNAs specific for the target miRNA and the RNA reporter. To enhance the CRISPR/crRNA applicability in synthetic biology projects, we developed the meta-CRISPR part collection in accordance with standardization, modularity and standard assembly rules. Our CRISPR collection allows the interchangeability of the genetic parts and the automation of construction following our standardized Golden Gate-based cloning strategy. Utilizing the meta-CRISPR collection, future iGEM teams can select the desired combination of DNA parts depending on the specific application of the CRISPR method.

Through the combination of different promoters and LbuCas13a coding sequences, one can select the desired LbuCas13a expression system, for example under the transcriptional control of the T7 or the pRha promoter. In addition, by combining the suitable genetic parts, researchers can select the purification method of the recombinant LbuCas13a protein either from inclusion bodies or from the soluble cytoplasmic fraction of the bacteria. Regarding the crRNA, utilizing the guidelines provided on the crPrep-crRNA preparation kit one can easily in silico design and produce the necessary crRNA sequence depending on the target miRNA utilizing the standardized cloning method and requiring only one additional primer. Last but not least, the LbuCas13a coding device can be assembled with the crRNA transcription system in a single plasmid enabling the simultaneous production of the LbuCas13a/crRNA complex in bacteria.

The parts contained in the meta-CRISPR collection are categorized into the following 3 subgroups:

• meta-CRISPR crRNA collection
• meta-CRISPR LbuCas13a collection
• meta-CRISPR LbuCas13a-crRNA combined collection

All parts, both basic and composite, included in our part collection have been successfully cloned into pSB1C3 shipping plasmid and are compatible with RFC [10] iGEM standard. DH5- alpha E.coli and BL21 (DE3) E.coli strains were used for routine cloning applications and protein expression, respectively. An overview of the meta-CRISPR collection and the interchangeable parts are described in Figure 1 and in table 1.

meta-CRISPR crRNA subcollection

The crRNA collection contains different crRNAs as designed by our dry lab team, targeting miR-17-3p and miR-17-5p through the DIAS detection platform. According to the modeling results, each crRNA demonstrates different analytical performance characteristics in detecting the above-mentioned miRNAs. To facilitate the easy implementation of the CRISPR/Cas13a system by all researchers even if they do not have significant expertise in designing in silico genetic sequences, we developed a methodology to produce the desired crRNA based on the target miRNA in an easy, efficient and low-cost procedure. In addition, this methodology facilitates the effortless alteration of the miRNA target of the DIAS platform extending the system's applications to different lung cancer-related miRNAs and even to other miRNA-related diseases. The guidelines provided on crPrep-crRNA preparation kit section. cover the whole range of crRNA-related experimental procedures starting from the in silico design, continuing with the experimental cloning in pSB1C3 plasmid and resulting in IV transcription and purification. This methodology allows the straightforward adjustment of the crRNA sequence depending on the targeted miRNA, modifying a specific primer and following the procedures provided on crPrep kit. The crRNA sequences are located downstream of the T7 promoter to allow for efficient in vitro transcription with T7 RNA polymerase (Moll et al., 2004).

meta-CRISPR LbuCas13a subcollection

The meta-CRISPR LbuCas13a subcollection contains all genetic parts related to the LbuCas13a. The CDS of LbuCas13a protein has been fragmented into 4 subparts (Cas13a part 1-4) which are flanked by BsaI type IIS restriction sites at both ends facilitating the easy assembly of the LbuCas13a subparts with the desired biobrick plasmid of the registry. This “fragmentation” simplifies the cloning procedures, allows for the easy synthesis of the required DNA sequences reducing complexity, permits the addition of the desired purification (His tag) or solubility tag (SUMO, MBP) modifying only the nucleotide sequence of the part 1 and facilitates bioengineering.

In addition, our LbuCas13a collection includes the LbuCas13a CDS sequence in fusion with the 6X His affinity purification tag as a single CDS sequence or with the addition of the SUMO solubility tag to allow for efficient protein production in the soluble cytoplasmic fraction (Butt et al., 2005). The internal restriction sites which are incompatible with the RFC [10] iGEM standard have been effectively removed with mutagenesis PCR.

In addition, our composite part collection includes the above-mentioned LbuCas13a sequences under transcriptional regulation of the T7 or the L-rhamnose-inducible promoter (pRha-BBa_K914003) to succeed transcriptional regulation with different systems and reagents (LacI & L-rhamnose). T7 polymerase systems enable a higher level of recombinant protein expression, however, they are prone to leaky expression complicating the strict control of the expression. On the other hand, the pRha-based systems provide tight transcriptional regulation without leaking but it produces a relatively small amount of recombinant protein. Depending on the downstream application of CRISPR/Cas13a system, researchers can select the tight transcriptional regulation of the LbuCas13a protein with the pRha promoter or the high-level, but still not tightly regulated, protein expression of the LbuCas13a protein with the T7 promoter (Giacalone et al., 2006).

meta-CRISPR LbuCas13a-crRNA combined subcollection

The meta-CRISPR LbuCas13a-crRNA combined collection contains only composite parts combining the crRNA sequences from the crRNA collection with the SUMO-LbuCas13a CDS from the LbuCas13a collection. According to literature references, the crRNA stability is significantly enhanced when it binds to Cas13a enzyme forming the Cas13-crRNA complex (Kick et al., 2022). The crRNAs after reconstitution with water should be stored at -80 deep freeze conditions and is prone to RNases activity. During the crRNA-related production and purification procedure, a significant amount of crRNA can be lost due to ribonuclease activity or degradation. Wanting to investigate this problem, we decided to clone the LbuCas13a coding sequence and the crRNA sequences into the same plasmid backbone (pSB1C3) and observe the simultaneous production of both the LbuCas13a protein in complex with the crRNA in E. coli cells. However, due to time limitations and iGEM competition requirements, these experiments have not yet been completed.