Table 1.
Efficiencies of pop-in, pop-out allele replacement in fission yeast.
| Allele | Modification | Pop-in homologous recombination a | Pop-out homologous recombination b | ||
|---|---|---|---|---|---|
| Homology L + R (bp) | Efficiency (H/T) | Homology L + R (bp) | Efficiency (M/T) | ||
| rec12-R76A | 2 bp substitutions | 937 + 676 | 7/8 (88%) | 225 + 1386 | 3/16(19%) |
| rec12-D79A | 2 bp substitutions | 937 + 676 | 7/8 (88%) | 231 + 1377 | 2/16 (13%) |
| rec12-E83A | 1 bp substitution | 937 + 676 | 7/8 (88%) | 247 + 1365 | 2/8 (25%) |
| rec12-R94A | 3 bp substitutions | 937 + 676 | 8/8 (100%) | 279 + 1331 | 3/16 (19%) |
| rec12-D95A | 3 bp substitutions | 937 + 676 | 7/8 (88%) | 337 + 1271 | 2/8 (25%) |
| rec12-Y97F | 2 bp substitutions | 937 + 676 | 3/3(100%) | 345 + 1265 | 3/8 (38%) |
| rec12-E179A | 2 bp substitutions | 209 + 1404 | 3/3 (100%) | 904 + 707 | 7/16 (44%) |
| rec12-K201A | 2 bp substitutions | 209 + 1404 | 3/3 (100%) | 770 + 841 | 9/12 (75%) |
| rec12-R209A | 4 bp substitutions | 209 + 1404 | 3/3 (100%) | 745 + 864 | 6/8 (75%) |
| rec12-K210A | 3 bp substitutions | 209 + 1404 | 3/3 (100%) | 743 + 865 | 6/8 (75%) |
| rec12-K214A | 4 bp substitutions | 209 + 1404 | 3/3 (100%) | 730 + 879 | 6/8 (75%) |
| rec12-D229A | 3 bp substitutions | 209 + 1404 | 2/2 (100%) | 685 + 924 | 6/16(38%) |
| rec12-D231A | 1 bp substitution | 209 + 1404 | 2/2 (100%) | 680 + 932 | 3/8 (38%) |
| rec12-K242A | 2 bp substitutions | 209 + 1404 | 2/2 (100%) | 647 + 964 | 5/8 (63%) |
| rec12-K282A | 4 bp substitutions | 937 + 676 | 2/2 (100%) | 526 + 1083 | 10/15 (67%) |
| rec12-R283A | 4 bp substitutions | 937 + 676 | 2/2 (100%) | 523 + 1086 | 5/12 (42%) |
| rec12-D284A | 3 bp substitutions | 937 + 676 | 2/2 (100%) | 521 + 1087 | 2/9 (22%) |
| rec12-R304A | 4 bp substitutions | 937 + 676 | 2/2 (100%) | 460 + 1149 | 3/8 (38%) |
| rec12-E305A | 1 bp substitution | 937 + 676 | 2/2 (100%) | 458 + 1154 | 4/26 (15%) |
| ade6-K87stop | 1 bp substitutions | 2012 + 854 | 8/10 c (80%) | 1134 + 1731 | 46/107 (43%) |
| ade6-Gal4BS | 7 bp substitutions | 2012 + 854 | 5/10 c(50%) | 1134 + 1705 | 25/67 (37%) |
| ade6- Gal4Control | 7 bp substitutions | 2012 + 854 | 8/10 c(80%) | 1134 + 1702 | 41/104 (39%) |
| ade6-rib+-M26 | 57 bp insertion | 1756 + 1110 | 5/6 (83%) | 899 + 1854 | 3/10 (30%) |
| ade6-ribm-M26 | 57 bp insertion | 1756 + 1110 | 4/6 (67%) | 899 + 1854 | 7/19 (37%) |
| ade6-rib+- M375 | 57 bp insertion | 1756 + 1110 | 3/6 (50%) | 899 + 1857 | 12/25 (48%) |
| ade6-ribm- M375 | 57 bp insertion | 1756 + 1110 | 2/3 (67%) | 899 + 1857 | 8/14 (57%) |
| ade6-M26-rib+ | 55 bp insertion | 2012 + 854 | 3/6 c (50%) | 1011 + 1720 | 27/94 (29%) |
| ade6-M26-ribm | 55 bp insertion | 2012 + 854 | 5/6 c (83%) | 1011 + 1720 | 33/100 (33%) |
| ade6-M375- rib+ | 55 bp insertion | 2012 + 854 | 4/6 c (67%) | 1008 + 1720 | 24/80 (30%) |
| ade6-M375- ribm | 55 bp insertion | 2012 + 854 | 5/6 c (83%) | 1008 + 1720 | 24/82 (29%) |
| ade6-M26- DP2 | 84 bp deletion | 1928 + 854 | 3/6 c (50%) | 441 + 1854 | 23/65 (35%) |
| ade6-M26- DP7 | 64 bp deletion | 1928 + 854 | 5/8 c (83%) | 750 + 1854 | 58/122 (48%) |
| prl10-rib+ | 57 bp insertion | 1557 + 383 | 6/7 (86%) | 1299 + 641 | 14/30 (47%) |
| prl10-ribm | 57 bp insertion | 1557 + 383 | 6/8 (75%) | 1299 + 641 | 14/30 (47%) |
| prl34-rib+ | 57 bp insertion | 1251 + 334 | 5/7 (71%) | 677 + 908 | 23/30 (77%) |
| prl34-ribm | 57 bp insertion | 1251 + 334 | 6/7 (86%) | 677 + 908 | 16/30 (53%) |
| fbp1- ΔM26 | 2 bp substitutions | 235 + 775 | 29/30 (97%) | 478 + 528 | 8/16 (50%) |
| ctt1- ΔM26 | 3 bp substitutions | 411 + 409 | 16/16 (100%) | 266 + 550 | 0/38 d (0%) |
| ctt1- ΔM26 | 3 bp substitutions | 411 + 409 | 5/20 (25%) | 266 + 550 | 3/16 (18%) |
| leu1-D1::prA- lexA | 1080 bp replacement | 939 + 1090 | 5/14 (36%) | 741 + 1288 | 3/12 (25%) |
Homology lengths are for regions to the left (L) and right (R) of the DSB used to promote recombination during transformation (Fig. 2a). The efficiency is the fraction of stable Ura+ transformants arising from pop-in homologous recombination (H) with the desired genomic target, as opposed to non-homologous integration elsewhere in the genome (T, total genotyped). For targeting constructs with insertions, the inserted DNA is not included in the lengths of homology.
Homology lengths flanking the desired modification are ordered relative to the schematic diagram in Fig. 2c–2d, where excision by recombination to the “left” (L) leaves the modification in the genome and excision to the “right” (R) leaves the wild-type allele in the genome. The efficiency is the fraction of pop-out recombinants that leave the modified allele (M) in the genome, as opposed to leaving the wild-type allele in the genome (T, total genotyped). Homology lengths do not include the region modified (e.g., where 3 base pairs were substituted within a 5 base pair stretch), so total lengths do not match precisely those reported for pop-in events.
In these cases we skipped genotyping of Ura+ transformants, picked 6–10 independent Ura+ colonies, sent them through pop-out protocols, and genotyped multiple FOAr derivatives of each clone.
In this unusual case, the Ura+ transformant had a tandem duplication of the target locus flanking the ura4+ cassette (desired structure), but after pop-out protocols never left a modified allele in the genome. We subsequently determined that both copies of the tandem integrant were wild-type. A different Ura+ transformant, with one wild-type and one modified allele in the tandem integrant, yielded the expected products (next line of table).