Table 2.
Numbers of called variants, showing total calls, which include all called variants compared to the reference including variants already present in the base strain, versus candidate de novo mutations (DNMs), which only include filtered variants likely to have happened during or after transformation of the platform strain. DNMs were further separated into on-target and off-target mutations. SnpEff off-target calls are reduced to variants classified by SnpEff to be within an open reading frame. Potential CRISPR/Cas9 off-target sites include sites with up to 10 mismatches from the used gRNA. The colony type describes if the sequencing was performed on a single colony or on a mixture of 5 colonies. The base strains relate to BSYBG10_3S1K_CalB, BSYBG10_chr3ne_HygR, and UPP-C (3S1K_CalB, chr3n3_HygR and UPP-C) and “#colonies” denotes the total number of colonies included in each group.
| Colony Type | CRIPR Plasmid | Base Strain (#Colonies) |
Type | Total Calls |
DNMs | DNMs On-Target |
DNMs Off-Target |
SnpEff Off-Target |
CRISPR Off-Target * |
|---|---|---|---|---|---|---|---|---|---|
| single | no gRNA | 3S1K_CalB (1) |
SNPs | 16 | 0 | - | 0 | 0 | - |
| InDels | 65 | 0 | - | 0 | 0 | - | |||
| SVs | 93 | 0 | - | 0 | - | - | |||
| single gRNA | UPP-C (6) |
SNPs | 91 | 1 | 0 | 1 | 1 | 0 | |
| InDels | 367 | 9 | 6 | 3 | 0 | 1 | |||
| SVs | 224 | 0 | 0 | 0 | - | 0 | |||
| 3S1K_CalB (3) |
SNPs | 52 | 2 | 0 | 2 | 2 | 0 | ||
| InDels | 188 | 3 | 2 | 1 | 1 | 0 | |||
| SVs | 190 | 1 | 1 | 0 | - | 0 | |||
| two gRNAs | 3S1K_CalB (9) |
SNPs | 161 | 0 | 0 | 0 | 0 | 0 | |
| InDels | 661 | 4 | 4 | 0 | 0 | 0 | |||
| SVs | 733 | 9 | 8 | 1 | - | 0 | |||
| Chr3ne_HygR (2) |
SNPs | 33 | 0 | 0 | 0 | 0 | 0 | ||
| InDels | 131 | 1 | 1 | 0 | 0 | 0 | |||
| SVs | 354 | 3 | 1 | 2 | - | 0 | |||
| mixed | no gRNA | 3S1K_CalB (10) |
SNPs | 268 | 1 | - | 1 | 0 | - |
| InDels | 209 | 1 | - | 1 | 1 | - | |||
| SVs | 284 | 0 | - | 0 | - | - | |||
| single gRNA | 3S1K_CalB (50) |
SNPs | 1935 | 10 | 0 | 10 | 4 | 1 | |
| InDels | 1133 | 31 | 23 | 8 | 6 | 2 | |||
| SVs | 1785 | 18 | 10 | 8 | 1 | ||||
| two gRNAs | 3S1K_CalB (30) |
SNPs | 898 | 10 | 0 | 10 | 4 | 3 | |
| InDels | 721 | 34 | 12 | 22 | 19 | 1 | |||
| SVs | 933 | 21 | 15 | 6 | - | 2 | |||
| Chr3ne_HygR (35) |
SNPs | 2776 | 24 | 1 | 23 | 16 | 2 | ||
| InDels | 698 | 28 | 16 | 12 | 5 | 3 | |||
| SVs | 1870 | 24 | 18 | 6 | - | 5 |
* All potential binding sites identified next to DNMs differed by at least 8 bases from the used gRNA.