RNA

Part:BBa_K2961005:Design

Designed by: Muhammad Nahin Khan   Group: iGEM19_CMUQ   (2019-10-16)


gRNA for Cystic Fibrosis Wildtype gene targeting of Cas12a / Cpf1


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 18
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

One must ensure that the target sequence does not contain the Cas12a PAM sequence, but rather that in the human genome, the PAM sequence of Cas12a is just upstream of it. (The PAM sequence for Cas12a is TTTN)


Source

The fixed loop part was adapted from the NEB website, as per their instructions (https://international.neb.com/faqs/2018/05/03/how-do-i-design-a-guide-rna-for-use-with-engen-lba-cas12a). The segment that is specific to the CFTR gene wild type variant was generated through CHOPCHOP by Harvard, and verified through BLAST and other genome browsing softwares.

References