Difference between revisions of "Part:BBa K3843008:Design"

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===Design Notes===
 
===Design Notes===
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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.
+
To ensure binding specificity for the Cas13a variant from <i>Leptotrichia buccalis</i>, 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 (CITE), 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 recognition sequence was adapted from Tovo-Rodrigues et al. (2013), 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.
 
The sequence was then converted to DNA (converting all U bases to T) in order to be submitted to the Parts Registry.
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===References===
 
===References===
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 +
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. <i>Cell. 184</i>(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. <i>PLoS ONE, 8</i>(12): e85164. https://doi.org/10.1371/journal.pone.0085164

Latest revision as of 20:56, 18 October 2021


LbuCas13a guide RNA - DRD4-targeting (missense deletion in VNTR motif 1)


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 Tovo-Rodrigues et al. (2013), 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 DRD4-mutant-targeting recognition sequence of the guide RNA was obtained from Tovo-Rodrigues et al. (2013): https://doi.org/10.1371/journal.pone.0085164

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