Table 1 Summary of two-step Nested CRISPR experiments
ExperimentPlates with dpy-10 edits/injected wormsPositives/worms screened (%)aIn-frame sequencesbCas9 concentration (ng/μl)
prpf-4::EGFP Step 11/136/23 (26.09)2 of 21500
prpf-4::EGFP Step 15/2428/50 (56.00)3 of 3250
prpf-4::EGFP Step 27/2313/32 (40.63)All1640
prpf-4::EGFP Step 2 synthetic sgRNA4/202/24 (8.33)cN/A250
prpf-4::mCherry Step 16/123/23 (13.04)2 of 3250
prpf-4::mCherry Step 211/2816/43 (37.21)All250
prpf-4::mCherry Step 2 synthetic sgRNA7/2826/108 (24.07)All250
prpf-4::2xTy1::EGFP::3xFLAG Step 113/2129/32 (90.63)3 of 3250
prpf-4::2xTy1::EGFP::3xFLAG Step 213/361/164 (0.61)All250
gtbp-1::EGFP Step 19/143/60 (5.00)d1 of 31000
gtbp-1::EGFP Step 13/737/52 (71.15)2 of 3250
gtbp-1::EGFP Step 29/2410/46 (21.74)All250
gtbp-1::EGFP Step 2 synthetic sgRNA4/133/34 (8.82)All250
gtbp-1::mCherry Step 16/1522/32 (68.75)1 of 3250
gtbp-1::mCherry Step 27/187/62 (11.29)All250
pgl-1::EGFP Step 15/247/50 (14.00)e1 of 11000
pgl-1::EGFP Step 14/1325/40 (62.50)2 of 5250
pgl-1::EGFP Step 25/161/13 (7.69)All250
pgl-1::mCherry Step 13/1324/45 (53.33)2 of 5250
pgl-1::mCherry Step 24/1814/39 (35.89)All250
K12C11.3p::mCherry Step 17/3721/56 (37.50)3 of 3250
K12C11.3p::mCherry Step 24/143/35 (8.57)All250
ubh-4::EGFP Step 12/88/30 (26.67)e1 of 1500
ubh-4::EGFP Step 29/274/39 (10.26)cAll250
mCherry::sftb-1 Step 12/425/29 (86.21)3 of 3250
mCherry::sftb-1 Step 22/231/3 (33.33)All250
EGFP::nfki-1 Step 23/122/26 (7.69)All250
  • a Based on PCR genotyping (amplicons of the correct size are considered positives regardless of whether or not the insertion is in-frame).

  • b In-frame sequences represent the fraction of worms with correct inserts over the PCR positives that were confirmed by Sanger sequencing (step 1). In step 2, all the worms that had PCR products of the correct size exhibited fluorescence, indicating in-frame insertions.

  • c Identification of positives via visual screening only.

  • d Pools of two to three worms.

  • e Pools of two worms. The percentage of positive events in worms screened in pools may be underestimated if two or more animals in the same pool are positive.