. 2018 Nov 7;8:16499. doi: 10.1038/s41598-018-34738-4

Table 3.

Total injected embryos, number and percent of dead and alive embryos, mean time to death (days post fertilization, dpf) ± standard error (SEM), mean time to hatch (dpf) ± SEM, hatch % ± SEM, fry survival % ± SEM and mean fry survival time (dpf) ± SEM for channel catfish (Ictalurus punctatus) embryos injected with different dosages of guide RNAs (gRNAs) and Cas9 protein targeting the channel catfish toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene.

Group	Total injected embryos	Dead embryos		Alive embryos		Embryo mean time to death (dpf) ± SEM	Mean time to hatch (dpf) ± SEM	Hatch % ± SEM	Fry survival % ± SEM	Mean fry survival time (dpf) ± SEM
Group	Total injected embryos	Number	%	Number	%	Embryo mean time to death (dpf) ± SEM	Mean time to hatch (dpf) ± SEM	Hatch % ± SEM	Fry survival % ± SEM	Mean fry survival time (dpf) ± SEM
TICAM 1
Low	140	63	45.0	77	55.0	6.0 ± 0.22^a	7.0 ± 0.08^a	55.0 ± 13.63^ab	70.1 ± 6.99 ^ab	15.2 ± 0.49^a
Medium	126	100	79.4	26	20.6	4.2 ± 0.21^b	7.4 ± 0.13^a	20.6 ± 9.65^a	53.8 ± 9.35 ^ab	14.3 ± 0.81^a
High	108	88	81.5	20	18.5	4.3 ± 0.24^b	7.3 ± 0.18^a	18.5 ± 5.71^a	80.0 ± 19.90 ^ab	16.2 ± 0.83^a
iCTRL	159	83	52.2	76	47.8	5.9 ± 0.18^a	7.1 ± 0.10^a	47.8 ± 4.85^ab	67.1 ± 1.51 ^a	15.1 ± 0.49^a
nCTRL	263	60	22.8	203	77.2	7.4 ± 0.08	7.1 ± 0.05^a	77.2 ± 7.60^b	96.6 ± 1.31 ^b	17.7 ± 0.13
RBL
Low	166	120	72.2	46	27.7	4.6 ± 0.18 ^a	6.3 ± 0.07^a	27.7 ± 2.57	80.4 ± 7.06	16.2 ± 0.56^a
Medium	124	104	83.9	20	16.1	4.2 ± 0.22 ^a	6.5 ± 0.12^ab	16.1 ± 0.75 ^a	75.0 ± 12.37	16.0 ± 0.78^a
High	173	145	83.8	28	16.2	5.0 ± 0.15 ^a	6.6 ± 0.10^b	16.2 ± 1.27 ^a	62.1 ± 2.75	14.8 ± 0.78^a
iCTRL	114	34	29.8	80	70.2	5.9 ± 0.17	6.1 ± 0.03^c	70.2 ± 0.90	97.5 ± 1.27	17.8 ± 0.12^b
nCTRL	244	32	13.1	212	86.9	6.7 ± 0.07	6.1 ± 0.02^c	86.9 ± 4.25	93.4 ± 3.39	17.4 ± 0.15^b
Combined TICAM1 and RBL
Low	306	183	59.8^b	123	39.8	5.4 ± 0.15	6.7 ± 0.06^b	40.2 ± 8.68^b	74.0 ± 4.82^a	15.6 ± 0.37^ab
Medium	250	204	81.6^a	46	18.4	4.3 ± 0.16^a	7.0 ± 0.11^a	18.4 ± 4.55^a	63.0 ± 7.75^a	15.0 ± 0.58^a
High	281	232	82.6^a	49	17.4	4.8 ± 0.14^a	6.9 ± 0.10 ^ab	17.4 ± 2.66^a	69.4 ± 9.25^a	15.3 ± 0.58^a
iCTRL	273	117	42.9^b	156	57.1	6.2 ± 0.14	6.6 ± 0.06^bc	57.1 ± 5.48^b	82.7 ± 6.78^a	16.5 ± 0.27^b
nCTRL	507	92	18.1	415	81.9	7.5 ± 0.06	6.5 ± 0.04^c	81.9 ± 4.46	94.9 ± 1.80^a	17.6 ± 0.10

Three dosages of gRNA were injected including low dosage (2.5 ng gRNAs, 7.5 ng Cas9 protein), medium dosage (5 ng gRNAs, 15 ng Cas9 protein) and high dosage (7.5 ng gRNAs, 22.5 ng Cas9 protein). Two control groups included a control that has been injected with the same volume of injection solution (composition: water with up to one third of 0.5% phenol red solution, no gRNA/Cas9 protein) as the treatment groups which was 50 nanoliters for all microinjected embryos (iCTRL). The second control group was not injected (nCTRL). Significant differences for means in the same column are marked with different superscript letters. Log Rank (Mantel-Cox) test was used for pairwise comparisons of embryo mean time to death, mean time to hatch, and mean fry survival time. One-way ANOVA was used to compare hatch percent and fry survival percent for different groups. When the equality of variance assumption was not satisfied, Welch’s test and Games-Howell test were performed. Significance level used for all tests was p = 0.05. Means in the same column with the same superscript letters are not significantly different (p > 0.05).