Part:BBa_K3843007:Design

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

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]

Design Notes

To ensure binding specificity for the Cas13a variant from Leptotrichia buccalis, the stem region was adapted from Fozouni et al. (2020), which used the same stem region successfully when designing guide RNAs for the detection of SAR-CoV-2 RNA.

The recognition sequence was adapted from Barr et al. (2000), where a 34-bp region containing the ADHD-associated mutation of interest was selected. A length of 34 bp was chosen specifically in order to ensure that the overall guide RNA sequence would be 64 bp, which is the recommended length for CRISPR-Cas13a.

The sequence was then converted to DNA (converting all U bases to T) in order to be submitted to the Parts Registry.

Source

The stem (hairpin stem+loop region) of the guide RNA was obtained from Fozouni et al. (2020): https://www.sciencedirect.com/science/article/abs/pii/S0092867420316238

The SNAP25-mutant-targeting recognition sequence of the guide RNA was obtained from Barr et al. (2000):

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.