Part:BBa_K3843007

LbuCas13a guide RNA - SNAP25-targeting (SNP T1065T>G)

CRISPR Cas systems involve a Cas protein and a CRISPR guide RNA; the Cas protein exhibits endonuclease activity when bound to its guide RNA. CRISPR-Cas13, specifically, cleaves a target single-stranded RNA (ssRNA) and remains bound. Once bound to the target, CRISPR-Cas13 will also exhibit nonspecific nuclease activity, indiscriminately cleaving ssRNA other than the target.

Fozouni et al. (2020) utilized Cas13a from Leptotrichia buccalis to detect SAR-CoV-2 RNA, favouring this species of Leptotrichia as it had the highest sensitivity compared to other Leptotrichia-derived Cas13a variants. Notably, this diagnostic method did not require prior amplification of the target RNA. Inspired by this, Waterloo iGEM 2021 adapted Cas13a from L. buccalis (henceforth referred to as LbuCas13a) for the detection of ADHD-associated mRNA transcripts in a diagnostic microfluidic assay.

In this case, the ADHD-associated gene chosen was SNAP25, which codes for a presynaptic membrane protein involved in the regulation of neurotransmitter release. The single nucleotide polymorphism (SNP) T1065T>G is known to be ADHD-associated (Faraone & Mick, 2010). As a result, this guide RNA was designed to target (and thereby detect) this mutation. When CRISPR-Cas13 binds this target and exhibits nonspecific nuclease activity, an ssRNA linker between a fluorophore and a quencher is cleaved, producing fluorescence.

References

Barr, C. L., Feng, Y., Wigg, K., Bloom, S., Roberts, W., Malone, M., Schachar, R., Tannock, R., & Kennedy, J. L. (2000, June 30). Identification of DNA variants in the snap-25 gene and linkage study of these polymorphisms and attention-deficit hyperactivity disorder. Molecular Psychiatry, 5, 405–409. https://www.nature.com/articles/4000733.

Faraone, S. V., & Mick, E. (2010, February 11). Molecular genetics of attention deficit hyperactivity disorder. Psychiatric Clinics of North America, 33(1), 159-180. https://www.sciencedirect.com/science/article/abs/pii/S0193953X09001063?via%3Dihub.

Fozouni, P., Son, S., Derby, M. D. de L., Knott, G. J., Gray, C. N., D’Ambrosio, M. V., Zhao, C., Switz, N. A., Kumar, G. R., Stephens, S. I., Boehm, D., Tsou, C.-L., Shu, J., Bhuiya, A., Armstrong, M., Harris, A. R., Chen, P.-Y., Osterloh, J. M., & Ott, M. (2020, December 4). Amplification-free detection of SARS-COV-2 with CRISPR-CAS13a and mobile phone microscopy. Cell. 184(2), 323-333.e9. https://www.sciencedirect.com/science/article/abs/pii/S0092867420316238.

Sequence and Features

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]