Part:BBa_K5087016

T7-3G IVT primer

Introduction 

T7-3G IVT primer is an IVT (In Vitro Transcription) primer specifically designed to anneal to the DNA template for crRNA production during IVT. This primer includes the sequence of the T7 promoter, enabling transcription. The resulting crRNA is then used in the SHERLOCK detection method [1].

Biology and Usage

The DNA template for crRNA must include a sequence complementary to T7-3G at its 3' end. During the annealing reaction, T7-3G binds to the template DNA strand at this complementary sequence, allowing the subsequent in vitro transcription (IVT) reaction to proceed.

IVT (In Vitro Transcription) [1]

The annealing reaction is initiated by a 5-minute denaturation step, followed by cooling to 4°C in a PCR thermocycler. This process results in the formation of a hybrid between the T7-3G primer and the ssDNA template.

In the subsequent IVT reaction, the T7 RNA polymerase recognizes the T7 promoter sequence present in the T7-3G primer and begins synthesizing RNA using the ssDNA template, producing crRNA.

Before using the crRNA in SHERLOCK assays, the IVT reaction mix must be purified by DNase treatment and chloroform/isopropanol extraction, or alternatively, with a specialized RNA purification kit.

Part Performance

Experimental details

Our team used the T7-3G promoter to produce the crRNAs for our tests using the traditional IVT method, prior to testing our SynLOCK system (BBa_K5087017). These crRNAs included SynCrRNA (BBa_K5087021), PrymCrRNA1 (BBa_K5087022), and PrymCrRNA2 (BBa_K5087023).

Annealing reaction

The annealing reaction was conducted following the protocol outlined by Kellner et al. [1] (steps 29-30).

Table 1. 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

2. 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

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

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

Table 3. IVT Reaction Mix components (for a single reaction) for PrymCrRNA1 and 2:

Component	Volume [µl]
H₂O	13
Annealing reaction mix	10
5x Buffer (from TranscriptAid T7 High Yield Transcription Kit, Thermo Scientific)	4
rNTP mix	10
Enzyme mix	2
Total	39

The reaction ran for 4 hours at 37°C. The samples were kept at -21°C until purification the next day.

Purification

Our chloroform/isopropanol extraction crRNA purification protocol is available here: RNA Purification Protocol

Results

Figure 1. Gel electrophoresis of the obtained IVT reaction product; 1 – RNA ladder, 2 – SynCrRNA 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.

Figure 2. Gel electrophoresis of the obtained IVT reaction product; 1 – RNA ladder, 2,3 – PrymcrRNA1, 4 – PrymcrRNA2 Conclusions: The products obtained appear to be the correct length when compared to previous electrophoresis results. The additional bands observed may be due to contamination or product degradation. Despite this, the samples were considered pure enough to proceed with further experiments.

Figure 3. Fluorescence readout results demonstrating the performance of various part combinations from our toolkit.

Conclusions:

• All crRNAs are functional. The annealing reaction, IVT, and purification processes were executed correctly. The T7-3G primer also worked properly, enabling successful IVT. 

• When the reaction was carried out with the appropriate target DNA (synthetic DNA from Kellner [1] for SynCrRNA and Prymnesium parvum DNA for PrymCrRNA1 and 2), strong fluorescence signals were observed. 

• In contrast, when a mismatch between the DNA template and crRNA was introduced (synDNA1 with PrymCrRNA1/2 and PrymDNA with SynCrRNA), no signal was detected, confirming the proper function of all crRNAs.

Sequence

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]

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 found in Kellner et al. article [1].

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