Part:BBa_K3843006

LbuCas13a guide RNA - DRD4-targeting (missense deletion in VNTR motif 3)

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 DRD4, which codes for a dopamine receptor. A 7-repeat allele containing a missense mutation has been shown to lead to a three-fold increase in the likelihood of hyperactivity (Tovo-Rodrigues et al., 2013). As a result, this guide RNA was designed to target one of these missense mutations within the 7-repeat region (specifically, a deletion in the variable tandem number repeat (VNTR) motif 3), allowing for the detection of 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

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.

Tovo-Rodrigues, L., Rohde, L. A., Menezes, A. M. B., Polanczyk, G. V., Kieling, C., Genro, J. P., Anselmi, L., & Hutz, M. H. (2013). DRD4 rare variants in attention-deficit/hyperactivity disorder (ADHD): Further evidence from a birth cohort study. PLoS ONE, 8(12): e85164. https://doi.org/10.1371/journal.pone.0085164

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]