Table 1.
Summary of sgRNA target sites for p300 binding area deletion.
| Name | Sequence | Genomic location (mm9) | Predicted cleavage efficiency | Off-target |
|---|---|---|---|---|
| mFurin-SE-sgRNA-L1 | caccGGAGGGCATGTCTTCGG CCTCCCGTACAGATAGCCcaaa |
Chr7: 87,561,784-87,561,801 | 65.62 | MM0 = 0, MM1 = 0, MM2 = 1, MM3 = 11 |
| mFurin-SE-sgRNA-L5 | caccGTGGGGGGAAGTACTCAT CACCCCCCTTCATGAGTAcaaa |
Chr7: 87,561,743-87,561,760 | 35.83 | MM0 = 0, MM1 = 1, MM2 = 6, MM3 = 116 |
| mFurin-SE-sgRNA-L11 | caccGGTGGAGGGCATGTCTAT CCACCTCCCGTACAGATAcaaa |
Chr7: 87,561,781-87,561,790 | 45.86 | MM0 = 0, MM1 = 0, MM2 = 8, MM3 = 27 |
| mFurin-SE-sgRNA-R1 | caccGGTCTTGGGATGTATCAC CCAGAACCCTACATAGTGcaaa |
Chr7: 87,562,511-87,562,528 | 54.66 | MM0 = 0, MM1 = 0, MM2 = 2, MM3 = 46 |
| mFurin-SE-sgRNA-R3 | caccgCTGTTGGTTTTGTAGGCT cGACAACCAAAACATCCGAcaaa |
Chr7: 87,562,529-87,562,546 | 32.67 | MM0 = 0, MM1 = 0, MM2 = 0, MM3 = 7 |
| mFurin-SE-sgRNA-R12 | caccgCCTATGGTCCCAGTGTTT cGGATACCAGGGTCACAAAcaaa |
Chr7: 87,562,423-87,562,439 | 54.48 | MM0 = 0, MM1 = 1, MM2 = 9, MM3 = 115 |
| mFurin-PRO-sgRNA-L1 | caccgCTGGTTCCTCCCAGATTG cGACCAAGGAGGGTCTAACcaaa |
Chr7: 87,548,976-87,548,995 | 51.23 | MM0 = 0, MM1 = 0, MM2 = 0, MM3 = 4 |
| mFurin-PRO-sgRNA-L2 | caccgTGGGGGCTTTCCCTTAGA cACCCCCGAAAGGGAATCTcaaa |
Chr7: 87,548,994-87,549,011 | 41.95 | MM0 = 0, MM1 = 0, MM2 = 0, MM3 = 10 |
| mFurin-PRO-sgRNA-L3 | caccgATGTCTGCAGTGTTTTAA cTACAACGTCACAAAATTcaaa |
Chr7: 87,548,945-87,548,962 | 41.95 | MM0 = 0, MM1 = 0, MM2 = 0, MM3 = 10 |
| mFurin-PRO-sgRNA-R1 | aaacCTCTCGAATCTGGATGGTc GAGAGCTTAGACCTACCAgccac |
Chr7: 87,549,484-87,549,500 | 71.91 | MM0 = 0, MM1 = 0, MM2 = 0, MM3 = 6 |
| mFurin-PRO-sgRNA-R2 | caccgACAGCATGTGTGTATAAA cTGTCGTACACACATATTTcaaa |
Chr7: 87,549,974-87,549,990 | 57.24 | MM0 = 0, MM1 = 0, MM2 = 2, MM3 = 43 |
| mFurin-PRO-sgRNA-R3 | caccgCAAGTGCTACCCACAATT cGTTCACGATGGGTGTTAAcaaa |
Chr7: 87,550,585-87,550,602 | 45.35 | MM0 = 0, MM1 = 0, MM2 = 0, MM3 = 8 |
One hundred fifty base pair sequences from both up- and downstream of the most abundant p300 binding regions in Furin promoter area (PRO) and 20 kb upstream Furin transcription site (super-enhancer area, SE) were analyzed for targeting with Cas9 endonuclease activity. Twenty sequences of each region were evaluated according to predicted Cas9 cleavage efficiency and off-target effect, and three sequences/region were chosen for experimental procedures. Upper case letters show the sequences in mouse genome, lower case letters refer to additional nucleotides for restriction enzyme digestion during cloning to the px330 vector. Predicted cleavage efficiency and off-target prediction are assessed using ChopChop (30). Off-target values show the number of off-targets with 0 (MM0), 1 (MM1), 2 (MM2), 3 or more (MM3) mismatches.