Thoughts on Using 2 Cas9 Nickases on to Make the DiNV BAC

Background: Through reading of the Addgene CRISPR handbook, the IDT Nickase webpage, and the IDT Nickase Application Note

  • There are two different mutations in the Cas9 protein, each produces an enzyme that only makes a 1 strand break
    • D10A only cleaves the target strand
    • H840A only cleaves the non-target strand
  • Need two guide RNAs, one for each strand/each Cas9. You need two Cas9s to make two cuts on the DNA to effectively create a DSB
  • The D10A Cas9 has a higher editing efficiency and works better to generate homology directed repair, so we should use that one
  • The orientation of sgRNAs that works best is “PAM-out”, where the PAM sites are on the outside of the two sgRNAs (see image)
  • The ideal number of bases between nick sites (so this is not between sgRNAs, but their specific nick sites) is 37-68bp

First I wanted to see if it was possible with our 4 regions of the DiNV genome to get 2 sgRNAs that have opposite orientation within these distances

77kb Region

  • 85 and 31
    • 85 cuts at 77,497
    • 31 cuts at 77,540
    • Nick distance is 43 bp
    • 85 activity score: 0.504
    • 31 activity score: 0.545
    • 0 off-tartget in CDS for either
  • 89 and 27
    • 89 cuts at 77,350
    • 27 cuts at 77,400
    • Nick distance is 50bp
    • 89 activity score: 0.351
    • 27 activity score: 0.716
    • 0 off-tartget in CDS for either

44kb Region

  • 58 and 1
    • 58 cuts at 44,346
    • 1 cuts at 44,395
    • Nick distance is 49bp
    • 58 activity score: 0.545
    • 1 activity score: 0.353
    • 7.31% off-target score for guide 1
  • 54 and 3
    • 54 cuts at 44,450
    • 3 cuts at 44,512
    • Nick distance is 62bp
    • 54 activity score: 0.662
    • 3 activity score: 0.516
    • 0 off-target in CDS for either
  • 44 and 11
    • 44 cuts at 44,882
    • 11 cuts at 44,948
    • Nick distance is 66bp
    • 44 activity score: 0.667
    • 11 activity score: 0.332
    • 0 off-target in CDS for either
  • 44 and 10
    • 44 cuts at 44,882
    • 10 cuts at 44,926
    • Nick distance is 44bp
    • 44 activity score: 0.667
    • 10 activity score: 0.340
    • 0 off-target in CDS for either
  • 32 and 14
    • 32 cuts at 45,061
    • 14 cuts at 45,119
    • Nick distance is 59bp
    • 32 activity score: 0.559
    • 14 activity score: 0.370
    • 0 off-target in CDS for either
  • 30 and 15
    • 30 cuts at 45,090
    • 15 cuts at 45,150
    • Nick distance is 60bp
    • 30 activity score: 0.589
    • 15 activity score: 0.509
    • 0 off-target in CDS for either
  • 26 and 18
    • 26 cuts at 45,267
    • 18 cuts at 45,332
    • Nick distance is 65bp
    • 26 activity score: 0.533
    • 18 activity score: 0.338
    • 0 off-target in CDS for either

75kb Region

  • 81 and 2
    • 81 cuts at 74,550
    • 2 cuts at 74,604
    • Nick distance is 54bp
    • 81 activity score: 0.586
    • 2 activity score: 0.555
    • 0 off-target in CDS for either
  • 81 and 1
    • 81 cuts at 74,550
    • 1 cuts at 74,603
    • Nick distance is 53bp
    • 81 activity score: 0.586
    • 1 activity score: 0.583
    • 0 off-target in CDS for either
  • 71 and 37
    • 71 cuts at 75,338
    • 37 cuts at 75,278
    • Nick distance is 55bp
    • 71 activity score: 0.572
    • 37 activity score: 0.578
    • 0 off-target in CDS for either
  • 68 and 42
    • 68 cuts at 75,338
    • 42 cuts at 75,393
    • Nick distance is 55bp
    • 68 activity score: 0.504
    • 42 activity score: 0.718
    • 0 off-target in CDS for either
  • 64 and 43
    • 64 cuts at 75,427
    • 43 cuts at 75,479
    • Nick distance: 52bp
    • 64 activity score: 0.662
    • 43 activity score: 0.509
    • 0 off-target in CDS for either
  • 57 and 51
    • 57 cuts at 75,540
    • 51 cuts at 75,593
    • Nick distance is 53bp
    • 57 activity score: 0.316
    • 51 activity score: 0.675
    • 0 off-target in CDS for either

130kb Region

  • 64 and 30
    • 64 cuts at 129,798
    • 30 cuts at 129,850
    • Nick distance is 52bp
    • 64 activity score: 0.552
    • 30 activity score: 0.589
    • 0 off-target in CDS for either
  • 9 and 28
    • 9 cuts at 130,765
    • 28 cuts at 130,835
    • Nick distance is 70bp
    • 9 activity score: 0.585
    • 28 activity score: 0.624
    • Both have off-target hits in the DiNV genome

I think it’s best not to pick any that have off-target hits. Or probably one of the guides with an activity score below 0.5. This leaves about 10 pairs of sgRNAs left.