Part:BBa_K2927012

DNA template of crRNA 3 for LbCas12a

This part encode the crRNA-3 that mediates Cas12a protein to recognize target sequences. Once crRNA binds with Cas12a protein, the complex capable to target and cleave targeted double-stranded DNA sequence (cis cleavage activity). The cleavage of double-stranded DNA activates the ability of Cas12a protein to cleave non-specific ssDNA (trans cleavage activity). In our project, crRNA-3 is designed to detect specific sequence of vp72 gene in African swine fever virus (ASFV).

Of note, crRNA-3 only contains 20 nucleotide sequences from vp72 gene of ASFV, which shows no potential of protein coding. Our design had been reported to iGEM and got approved.  

Experiment results of crRNA synthesis

To product our crRNA, we use PCR to replicate the template of crRNA.

Fig 1. Gel electrophoresis of PCR product of crRNA template.

As fig. 7 shows, we synthesis the band between 200 bp and 100 bp, which is the correct size of crRNA template length (161 bp). So we use gel extraction to purified crRNA template.

To complete our DNA template, we need KpnI restriction enzyme to cut down unnecessary sequence, to avoid in vitro transcription(IVT) to keep product unnecessary crRNA.

Fig 2. Gel electrophoresis of PCR product of crRNA template cut by KpnI.

Final, before IVT and RNA condensation, we run 1% agarose gel electrophoresis to check result.

Fig 3. Gel electrophoresis of crRNA.

The only different between two gels is the blank of brightness. As result shows in the pictures, our band is located between 200 bp and 100 bp. So we can make sure our crRNA product success.