Table 1.
Using ITR-Seq for on- and off-target evaluation of sgRNA-dependent nucleases
| Vector 1 (GC/kg dose) | Vector 2 (GC/kg dose) | Time point(days) | Indel% (on-target) | Off-Target Sites | Target locus | Target Sequence | ||
|---|---|---|---|---|---|---|---|---|
| Mouse A | Mouse B | Mouse A | Mouse B | |||||
| AAV8-SaCas9 (3 × 1011) | AAV8-sgRNA1 (2 × 1012) | 21 | 28.05 | 32.59 | 3 | 4 | ASS1 (chr2:31518639–31518658) | ACAGGACTCCCAGAGTTAGA |
| AAV8-LbCpf1 (3 × 1011) | AAV8-sgRNA1 (2 × 1012) | 21 | 14.35 | 23.87 | 1 | 2 | ASS1 (chr2:31518480–31518502) | CAAATGGCAGGAAGAATTCACGG |
| AAV8-LbCpf1 (3 × 1011) | AAV8-sgRNA2 (2 × 1012) | 21 | 33.42 | 27.64 | 4 | 3 | ASS1 (chr2:31519012–31519034) | TGGCTGGAAATATTAGGGCAACT |
| AAV8-AsCpf1 (3 × 1011) | AAV8-sgRNA1 (2 × 1012) | 21 | 1.22 | 0.38 | 3 | 1 | ASS1 (chr2:31518480–31518502) | CAAATGGCAGGAAGAATTCACGG |
| AAV8-AsCpf1 (3 × 1011) | AAV8-sgRNA2 (2 × 1012) | 21 | 0.28 | 0.12 | 2 | 1 | ASS1 (chr2:31519012–31519034) | TGGCTGGAAATATTAGGGCAACT |
| AAV8-SaCas9 (3 × 1011) | AAV8-sgRNA1 (1 × 1012) | 70 | 26.94 | 29.11 | 10 | 12 | ASS1 (chr2:31518639–31518658) | ACAGGACTCCCAGAGTTAGA |
| AAV8-SaCas9 (1 × 1011) | AAV8-sgRNA1 (1 × 1012) | 70 | 21.39 | 25.98 | 7 | 9 | ASS1 (chr2:31518639–31518658) | ACAGGACTCCCAGAGTTAGA |
| AAV8-LbCpf1 (1 × 1011) | AAV8-sgRNA2 (1 × 1012) | 70 | 4.91 | 4.90 | 5 | 6 | ASS1 (chr2:31519012–31519034) | TGGCTGGAAATATTAGGGCAACT |
| AAV8-LbCpf1 (3 × 1011) | AAV8-sgRNA2 (1 × 1012) | 70 | 7.69 | 8.48 | 10 | 4 | ASS1 (chr2:31519012–31519034) | TGGCTGGAAATATTAGGGCAACT |