Table 1. Summary of two-step Nested CRISPR experiments.
| Experiment | Plates with dpy-10 edits/injected worms | Positives/worms screened (%)a | In-frame sequencesb | Cas9 concentration (ng/μl) |
|---|---|---|---|---|
| prpf-4::EGFP Step 1 | 1/13 | 6/23 (26.09) | 2 of 2 | 1500 |
| prpf-4::EGFP Step 1 | 5/24 | 28/50 (56.00) | 3 of 3 | 250 |
| prpf-4::EGFP Step 2 | 7/23 | 13/32 (40.63) | All | 1640 |
| prpf-4::EGFP Step 2 synthetic sgRNA | 4/20 | 2/24 (8.33)c | N/A | 250 |
| prpf-4::mCherry Step 1 | 6/12 | 3/23 (13.04) | 2 of 3 | 250 |
| prpf-4::mCherry Step 2 | 11/28 | 16/43 (37.21) | All | 250 |
| prpf-4::mCherry Step 2 synthetic sgRNA | 7/28 | 26/108 (24.07) | All | 250 |
| prpf-4::2xTy1::EGFP::3xFLAG Step 1 | 13/21 | 29/32 (90.63) | 3 of 3 | 250 |
| prpf-4::2xTy1::EGFP::3xFLAG Step 2 | 13/36 | 1/164 (0.61) | All | 250 |
| gtbp-1::EGFP Step 1 | 9/14 | 3/60 (5.00)d | 1 of 3 | 1000 |
| gtbp-1::EGFP Step 1 | 3/7 | 37/52 (71.15) | 2 of 3 | 250 |
| gtbp-1::EGFP Step 2 | 9/24 | 10/46 (21.74) | All | 250 |
| gtbp-1::EGFP Step 2 synthetic sgRNA | 4/13 | 3/34 (8.82) | All | 250 |
| gtbp-1::mCherry Step 1 | 6/15 | 22/32 (68.75) | 1 of 3 | 250 |
| gtbp-1::mCherry Step 2 | 7/18 | 7/62 (11.29) | All | 250 |
| pgl-1::EGFP Step 1 | 5/24 | 7/50 (14.00)e | 1 of 1 | 1000 |
| pgl-1::EGFP Step 1 | 4/13 | 25/40 (62.50) | 2 of 5 | 250 |
| pgl-1::EGFP Step 2 | 5/16 | 1/13 (7.69) | All | 250 |
| pgl-1::mCherry Step 1 | 3/13 | 24/45 (53.33) | 2 of 5 | 250 |
| pgl-1::mCherry Step 2 | 4/18 | 14/39 (35.89) | All | 250 |
| K12C11.3p::mCherry Step 1 | 7/37 | 21/56 (37.50) | 3 of 3 | 250 |
| K12C11.3p::mCherry Step 2 | 4/14 | 3/35 (8.57) | All | 250 |
| ubh-4::EGFP Step 1 | 2/8 | 8/30 (26.67)e | 1 of 1 | 500 |
| ubh-4::EGFP Step 2 | 9/27 | 4/39 (10.26)c | All | 250 |
| mCherry::sftb-1 Step 1 | 2/4 | 25/29 (86.21) | 3 of 3 | 250 |
| mCherry::sftb-1 Step 2 | 2/23 | 1/3 (33.33) | All | 250 |
| EGFP::nfki-1 Step 2 | 3/12 | 2/26 (7.69) | All | 250 |
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.