Using EnGen® sgRNA Synthesis Kit, S. pyogenes (NEB #E3322) and Protocol To Design Oligos Needed for Making sgRNAs for DiNV

Need to design oligos with the 20 nucleotide guide sequence and the appropriate upstream and downstream sequences for the kit that we can then have synthesized for us.

  • Using the top 3 guide RNA sites generated from Geneious that are within the 77,000 and 78,000 region of the DiNV genome. These are #s 92, 27, and 86. However #27 seems to have an off target BLAST hit of the sgRNA sequence to the D. virilis genome. So we might not want to use that one. Guide RNA #73 has a lower activity score but no off target hits. Sequences from here
  • Guide RNA 92 sequence: 5’ AAAAAATGCACTAAAACACA, activity score: 0.719, reverse direction
  • Guide RNA 27 sequence: 5’ TGTATTGTCAGTGTGGGAGA, activity score: 0.716, forward direction
  • Guide RNA 86 sequence: 5’ ACACAAGTGTTGTTATACGA, activity score: 0.713, reverse direction
  • Guide RNA 73 sequence: 5’ ATCTAAATAAATACAATCGA, activity score: 0.623, reverse direction

Steps in synthesis kit protocol

  1. Target sequences should be 20nt long (yes), and they don’t include the PAM. PAM is on the 5’ end
  2. If there is not a G at the 5’ end of the sequences, add 1 (need to add to all because they all have an A)
    • 92: 5’ GAAAAAATGCACTAAAACACA
    • 27: 5’ GTGTATTGTCAGTGTGGGAGA
    • 86: 5’ GACACAAGTGTTGTTATACGA
    • 73: 5’ GATCTAAATAAATACAATCGA
  3. To the 5´ end add the T7 promoter sequence: TTCTAATACGACTCACTATA
    • 92: 5’ TTCTAATACGACTCACTATAGAAAAAATGCACTAAAACACA
    • 27: 5’ TTCTAATACGACTCACTATAGTGTATTGTCAGTGTGGGAGA
    • 86: 5’ TTCTAATACGACTCACTATAGACACAAGTGTTGTTATACGA
    • 73: 5’ TTCTAATACGACTCACTATAGATCTAAATAAATACAATCGA
  4. To the 3´ end add the 14 nucleotide overlap sequence: GTTTTAGAGCTAGA
    • 92: 5’ TTCTAATACGACTCACTATAGAAAAAATGCACTAAAACACAGTTTTAGAGCTAGA
    • 27: 5’ TTCTAATACGACTCACTATAGTGTATTGTCAGTGTGGGAGAGTTTTAGAGCTAGA
    • 86: 5’ TTCTAATACGACTCACTATAGACACAAGTGTTGTTATACGAGTTTTAGAGCTAGA
    • 73: 5’ TTCTAATACGACTCACTATAGATCTAAATAAATACAATCGAGTTTTAGAGCTAGA
  5. Check final sequence to make sure it’s copied right
    • 92: 5’ TTCTAATACGACTCACTATAGAAAAAATGCACTAAAACACAGTTTTAGAGCTAGA
    • 27: 5’ TTCTAATACGACTCACTATAGTGTATTGTCAGTGTGGGAGAGTTTTAGAGCTAGA
    • 86: 5’ TTCTAATACGACTCACTATAGACACAAGTGTTGTTATACGAGTTTTAGAGCTAGA
    • 73: 5’ TTCTAATACGACTCACTATAGATCTAAATAAATACAATCGAGTTTTAGAGCTAGA