Part:BBa_K5087021

SynCrRNA1 spacer template

Introduction

This part is the DNA template for the spacer sequence of SynCrRNA, used to design a crRNA molecule that targets the synthetic DNA sequence employed as a positive control in SHERLOCK assays. It serves to verify material quality and helps the experimenter become familiar with the SHERLOCK protocol. To ensure proper crRNA function, the DR (direct repeat) loop sequence specific to the Cas13 protein used in the assay must be included.

Biology [1]

CrRNA (CRISPR-RNA) is the molecule that guides Cas13 proteins of the SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking) platform to their specific targets, making it essential for accurate identification of the targeted sequence.

The crRNA consists of two key components: the DR loop and the spacer sequence. The DR loop is crucial for attaching the Cas13 protein to the crRNA molecule, while the spacer is a 28-30 nucleotide programmable sequence complementary to the detection target. It enables the Cas13 protein to be accurately guided to the target for precise identification of the target sequence.

Figure 1. Scheme of crRNA’s DNA template with appended T7 promoter and DR sequence (one method for crRNA production) used with the huLwCas13a.

The SHERLOCK method [1, 2]

The SHERLOCK platform is a modern synthetic biology tool that utilizes the properties of the Cas13 protein, an enzyme from the Nobel Prize-winning CRISPR-Cas system. The Cas13 protein is guided with high specificity to the target sequence using crRNA.

The crRNA molecule is crucial for the assay's specificity. It consists of a direct repeat (DR) sequence and a spacer sequence that is complementary to the target. The crRNA molecule is designed to uniquely identify the organism by targeting the Internal Transcribed Spacer (ITS) sequence in its genome.

First, the Cas13 protein binds to the organism's genetic material, which was previously amplified using RPA and transcribed into RNA. Once bound, the Cas13 protein is activated and exhibits a “collateral” RNase activity, meaning it non-specifically cleaves nearby single-stranded RNA molecules.

This activity can be used in assays by including synthetic RNA probes tagged with a fluorescent reporter and a quencher in the reaction mixture. A fluorescent signal indicates that the reporters have been cleaved by Cas13, confirming the presence of the DNA target in the sample. The SHERLOCK method can also be used with Lateral Flow Assays (LFA).

Figure 2. The SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) Method Mechanism.

Experimental validation

We obtained SynCrRNA molecules by both methods: using the traditional in vitro transcription by appending the T7 promoter to the crRNA template sequence (DR loop + spacer), as well as by incorporating the spacer of the SynCrRNA into the SynLOCK Cassette BBa_K5087017.

Traditional In Vitro Transcription (IVT)

For this reaction, a T7 promoter and a DR loop were appended to this part's sequence, resulting in the following template sequence (the whole template had to be ordered):

Table 1. crRNA Template Sequence for Reaction

5’-CTCGACCTGCAGGCATGCAAGCTTGGCGGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC-3’

An annealing reaction of the template with the T7-3G (BBa_K5087016) oligonucleotide was performed as suggested in Kellner et al. [1].

Table 2. Annealing Reaction Mix components (for a single reaction):

Component	Volume [µl]
crRNA template, 100 µM	1
T7-3G oligonucleotide, 100 µM	2
Standard Taq buffer, 10x	1
H₂O	7
Total	11

A 5-minute denaturation was conducted, after which the reaction was slowly cooled in a thermocycler to 4°C, at a rate of 0.1°C/s.

IVT reaction was performed. The reaction mix content was as follows:

Table 3. IVT Reaction Mix components (for a single reaction):

Component	Volume [µl]
H₂O	15
Annealing reaction mix	10
5x Buffer (from TranscriptAid T7 High Yield Transcription Kit, Thermo Scientific)	4
rNTP mix	8
Enzyme mix	2
Total	39

The reaction ran for 4 hours at 37°C. The samples were kept at -21°C until the next day. Then, the product was purified according to our protocol that can be found here.

We conducted RNA electrophoresis to check the length of the IVT product.

Results

Figure 3. Gel electrophoresis of the obtained IVT reaction product.

Conclusions: A clear and distinct RNA band was observed, confirming that the IVT reaction was successful. Although the RNA ladder did not develop correctly, this does not impact the interpretation of the results.

SynLOCK system IVT

As part of our contribution to the synthetic biology community, we developed a crRNA synthesis system that allows scientists to easily obtain crRNAs with custom spacers. The SynLOCK system enables the creation of crRNAs with a 28-nucleotide spacer sequence tailored to the user's needs.

• We directly cloned the spacer template into the SynLOCK Cassette BBa_K5087017. You can read more on how to use the system and how to design the spacers on the Cassette’s part page.

• We then linearized the isolated plasmids carrying the SynLOCK system with BbsI-HF enzyme (New England Biolabs) according to the provided manual.

The digestion reaction was then visualized by gel electrophoresis:

Figure 4. Gel Electrophoresis Results of the digestion of SynLOCK system plasmids carrying respective crRNA templates with BbsI. 1: Plasmid carrying PrymCrRNA1, 2: Plasmid carrying PrymCrRNA2, 3: Plasmid carrying SynCrRNA.

Conclusions: All plasmids were correctly linearized with BbsI, as indicated by a single band on the gel.

We then proceeded with IVT and post-IVT cleanup, according to our optimized protocol which can be accessed here.

The obtained concentrations of crRNAs were as described in Table 3. For more information on the SVR and USV plasmids used to carry our system, visit the SynLOCK Cassette part page.

Table 4. Concentrations of obtained SynCrRNA using SynLOCK system.

crRNA	Concentration [ng/μl]
SVR SyncrRNA	1637.9
USV SyncrRNA	742
FLuc Control Template*	672.2

*The control was provided with the HiScribe® T7 High Yield RNA Synthesis Kit (New England Biolabs) we used for this IVT reaction.

We conducted RNA electrophoresis to check the length of the IVT product.

Figure 5. RNA electrophoresis – SynLOCK system IVT results.

Conclusions: Transcripts of the correct length were successfully obtained.

Part Performance

The performance of this basic part is described on the SynCrRNA (BBa_K5087024) composite part page under the sections "Test: Experimental Validation" and "PrymFlow."

Sequence

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 7
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 7
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 7
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 7
  
• 1000
  COMPATIBLE WITH RFC[1000]

Biosafety

We used the Asimov's tool — Kernel — to check the sequence's safety with the Biosecurity Sequence Scanner. The results showed no flagged sequences, confirming that this part is safe to use.

Sequence source and Design

This sequence was introduced by Kellner et al. [1]. In the publication, it is referred to as “Synthetic DNA 1 LwaCas13a crRNA IVT template”.

Resources

• [1] Kellner, Max J., Jeremy G. Koob, Jonathan S. Gootenberg, Omar O. Abudayyeh, and Feng Zhang. “SHERLOCK: Nucleic Acid Detection with CRISPR Nucleases.” Nature Protocols 14, no. 10 (October 2019): 2986–3012. https://doi.org/10.1038/s41596-019-0210-2
• [2] Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science (1979). 2017 Apr 28;356(6336):438–42.