Table 1 Summary of experiments
ExperimentStrategy (Figure 1A)EditGenesgRNAsNo. of P0’saNo. 
of PCRsbNo. of F1’scNo. of
editsd% editseNo. of precise edits/no. of sequenced editsVerification of expression
Insertion of premature stop
11Insertion-STOPK08F4.2APa4-218277554417.45/5
21Insertion/deletion-STOPK08F4.2APs1395178137.32/2
31Insertion/deletion-STOPK08F4.2APs4614126710.4
41Insertion/deletion-STOPnos-2SL225; SL232319426731.13/3
Insertion of small protein tag
52TetraCys at C terminusK08F4.2APs61118837600.0
62TetraCys at C terminusK08F4.2APs5514328651.72/5In gel detection
72Myc, 3× Flag, V5, His, HA at C terminusK08F4.2APs5917931630.91/3WB/IF
82V5, 3× Flag, OLLAS at N terminusmex-5JS129; JS13011235412122.93/4f
91Myc, V5 at N terminusswan-1CSD3511919526.33/3WB/IF
1023× Flag at C terminuspgl-1TL001; TL002613413410.80/1
1123× Flag at N terminusnos-2SL225; SL23211370781192.46/8WB/IF
122V5 at N terminusmbk-2HS5162484812.11/1WB/IF
Insertion of GFP
131GFP at sgRNA site near C terminusK08F4.2APs512108741304.04/4GFP expression
142GFP at C terminusK08F4.2APs5201791134100.9GFP expression
152GFP at C terminusfbf-2APs12; APa131713483230.4GFP expression
161GFP at C terminusmes-2sg161815073850.7GFP expression
171GFP at C terminuslin-15bsg23; sg2555133620.6GFP expression
182GFP at C terminusdeps-1sg6; sg21118348771.4GFP expression
191GFP at C terminusmex-6sg3; sg18109066681.2GFP expression
202GFP at C terminusglh-1sg455440200.0
211GFP at C terminushtp-3sg51128400.0
Deletion
223ORF deletion (1.6 kb)K08F4.2(APs1; APs4); (APs5; APs6)16117675223.30/3
233ORF deletion (2.6 kb)mbk-2HS516; (JS117; JS118)116610832.8f
243Operon deletion (6 kb)swan-1/2CSD35; (CSD53; CSD54)118631851.60/2
254ORF deletion/NheI insertionK08F4.2APs1; APs51184528203.82/2
261ORF deletion/NheI insertionK08F4.2APs5157347500.0
  • WB, Western blot; IF, immunofluorescence; GFP expression, GFP fluorescence in live animals in a pattern expected for the targeted ORF (see Figure 3).

  • a Number of injected hermaphrodites whose broods were screened.

  • b Total number of F1 pools that were screened by PCR.

  • c Number of F1’s screened.

  • d Number of positive PCRs.

  • e Number of positive PCRs divided by the number of F1’s screened. The assumption is that each positive pool contains only one positive F1 animal. This may be an underestimate for the GFP experiments where each pool contained eight F1’s. Indeed, in some pools, we observed a higher frequency of edits among F2’s than expected if the pool contained only one edited F1.

  • f Edits are maternal-effect lethal (V5-tagged MEX-5 and deletion in mbk-2 locus). Interestingly, 2/2 independent V5-MEX-5 edits are maternal-effect lethal as is a mex-5(0) mutant (Schubert et al. 2000). In contrast, the FLAG-MEX-5 and OLLAS-MEX-5 edits were viable. The ability to generate fusions to different tags in the same experiment will be useful to determine the best tagging strategy for each gene and avoid those tags that interfere with normal protein function.