Table 3.
Efficiency of genome editing for other mutations.
| Gene | Strain | Induction | CRISPEY System | Edit | gRNA Scorea | Efficiency | Screening Method |
|---|---|---|---|---|---|---|---|
| ADE2 | BY4742 (JL4) | GAL, 48 h | pCas9-EcRT-Hyg, pCRISPEY-URA3 | Frameshift at position 466 | 0.00 | 100% (41/41) | red-white screening |
| ADE2 | BY4742 (JL1412) | GAL, 48 h | Integrated Cas9-EcRT, pCRISPEY-URA3 | Frameshift at position 472 | 1.34 | 100% (10/10) | Sanger sequencing |
| CAN1 | BY4742 (JL1412) | GAL, 48 h | Integrated Cas9-EcRT, pCRISPEY-URA3 | Frameshift at position 786 | 1.7 |
a100% (79/79) b100% (5/5) |
aPhenotype (canavanine resistance) bSanger sequencing |
| FLO8 | NCYC3449 (JL 998) | GAL, 72 h | pCas9-EcRT-Hyg, pCRISPEY-URA3 | Deletion between pos 161 and pos 310 of ORF; gRNA at pos 164 | 3.49 | 80% (8/10) | Phenotype (loss of flocculation) |
| HIS3 | I14 (JL 1809) | GAL, 72 h | pCas9-EcRT-Hyg, pCRISPEY-URA3 | Deletion between pos 308 and pos 497 of ORF; gRNA at pos 315 | 3.05 | 67% (10/15) | Phenotype (His auxotrophy) |
| HIS3 | NCYC3449 (JL1810) | GAL, 72 h | pCas9-EcRT-Hyg, pCRISPEY-URA3 | Deletion between pos 308 and pos 497 of ORF; gRNA at pos 315 | 3.05 | 53% (8/15) | Phenotype (His auxotrophy) |
| HIS3 | NCYC3462 (JL1811) | GAL, 72 h | pCas9-EcRT-Hyg, pCRISPEY-URA3 | Deletion between pos 308 and pos 497 of ORF; gRNA at pos 315 | 3.05 | 7% (2/15) | Phenotype (His auxotrophy) |
| HIS3 | DY9 (JL1812) | GAL, 72 h | pCas9-EcRT-Hyg, pCRISPEY-URA3 | Deletion between pos 308 and pos 497 of ORF; gRNA at pos 315 | 3.05 | 16% (4/25) | Phenotype (His auxotrophy) |
| HO | NCYC3449 (JL1811) | GAL, 72 h | pCas9-EcRT-Hyg, pCRISPEY-URA3 | Deletion between first and last 50 bp of ORF; gRNA at pos 1121 | 2.84 | 100% (5/5) | PCR |
| HO | NCYC3462 (JL1040) | GAL, 72 h | pCas9-EcRT-Hyg, pCRISPEY-URA3 | Deletion between first and last 50 bp of ORF; gRNA at pos 1121 | 2.84 | 75% (6/8) | PCR |
| HO | I14 (JL996) | GAL, 72 h | pCas9-EcRT-Hyg, pCRISPEY-URA3 | Deletion between first and last 50 bp of ORF; gRNA at pos 1121 | 2.84 | 63% (5/8) | PCR |
| HO | DY9 (JL1001) | GAL, 72 h | pCas9-EcRT-Hyg, pCRISPEY-URA3 | Deletion between first and last 50 bp of ORF; gRNA at pos 1121 | 2.84 | 67% (2/3) | PCR |
| LEU2 | NCYC3462 (JL1811) | GAL, 72 h | pCas9-EcRT-Hyg, pCRISPEY-URA3 | Deletion between pos 36 and pos 1095 of ORF; gRNA at pos 599 | 3.37 | 27% (4/15) | Phenotype (Leu auxotrophy) |
| LEU2 | I14 (JL1828) | GAL, 72 h | Integrated Cas9-EcRT, pCRISPEY-URA3 | Deletion between pos 36 and pos 1095 of ORF; gRNA at pos 599 | 3.37 | 100% (15/15) | Phenotype (Leu auxotrophy) |
| LEU2 | DY9 (JL1829) | GAL, 72 h | Integrated Cas9-EcRT, pCRISPEY-URA3 | Deletion between pos 36 and pos 1095 of ORF; gRNA at pos 599 | 3.37 | 100% (15/15) | Phenotype (Leu auxotrophy) |
| LEU2 | NCYC3462 (JL1830) | GAL, 72 h | Integrated Cas9-EcRT, pCRISPEY-URA3 | Deletion between pos 36 and pos 1095 of ORF; gRNA at pos 599 | 3.37 | 100% (15/15) | Phenotype (Leu auxotrophy) |
| LEU2 | NCYC3449 (JL1831) | GAL, 72 h | Integrated Cas9-EcRT, pCRISPEY-URA3 | Deletion between pos 36 and pos 1095 of ORF; gRNA at pos 599 | 3.37 | 100% (15/15) | Phenotype (Leu auxotrophy) |
| LEU2 | DBY8268 (JL1662) | GAL, 72 h | Integrated Cas9-EcRT, pCRISPEY-URA3 | Deletion between pos 36 and pos 1095 of ORF; gRNA at pos 599 | 3.37 | 89% (16/18) | Phenotype (Leu auxotrophy) |
| TPS1 | YPS163 (JL1673) | GAL, 72 h | pCas9-EcRT-HYG, NAT selection for CRISPEY gRNA | deletion of the TPS1 ORF, gRNA at pos 470 | −0.5 | 50% (5/10) | PCR |
| TPS1 | M22 (JL123) | GAL, 72 h | pCas9-EcRT-KAN, NAT selection for CRISPEY gRNA | deletion of the TPS1 ORF, gRNA at pos 470 | −0.5 | 22% (2/9) | PCR |
a
gRNA scores based on sgRNA Scorer 2 (Chari et al., 2017), with the following being the percentile ranks for predicted gRNA activities (from low to high activity): −2.59 (10th percentile ), 1.43 (25th), −0.14 (50th), 1.15 (75th), and 2.27 (10th).