Table 1. CRISPR mutational interrogation experiments at putative Ubx regulatory regions.

Species	sgRNA(s)	Inj. EmbryosNinj	L1 larvaeNhat	Pupae orL5 larvae	AdultsNadu	CrispantsNmut	Inj. timeh AEL	Cas9:sgRNA ng/µL	Hatching RateNhat/Ninj	Crispant RateNmut/Ninj
J. coenia	Antp-Ubx_BE	59	50	50	44	6	2.5–3.5	500 : 250	84.7%	10.2%
		118	40	40	31	6	1.75–2.75	250 : 125	33.9%	5.1%
		89	44	44	39 *	17	2.25–3.5	500 : 250	49.4%	19.1%
	Total	266	90	134	115	29			33.8%	10.9%
V. cardui	IT1_sgRNA1	204	67	50	50	2	1–3	250 : 125	32.8%	1.0%
		108	49	31	31	3	2–3	125 : 62.5	45.4%	2.8%
		145	60	39	39	2	2.25–3.5	500 : 250	41.4%	1.4%
	Total	457	176	120	120	7			38.5%	1.5%
J. coenia	IT1_sgRNA2	59	40	7	6	0	0.5–2.5	500 : 250	67.8%	0.0%
		124	112	112	110	4	2.25–4.75	500 : 250	90.3%	3.2%
	Total	183	152	119	116	4			83.1%	2.2%
V. cardui	AS5_sgRNA1	334	183	57	52	5	2–3	250 : 125	54.8%	1.5%
		122	87	2	2	0	2–4	500 : 250	71.3%	0.0%
	Total	456	270	59	54	5			59.2%	1.1%
J. coenia	AS5_sgRNA1	309	181	181	181	8	2–4.5	500 : 250	58.6%	2.6%
J. coenia	Ubx11a2+3 +c5+6	317	18	-	-	2	1–3	500 : 75 ea.	5.7%	0.6%
		203	35	0	0	0	1.5–3.5	500 : 75 ea.	17.2%	0.0%
	Total	520	53	-	-	2			10.2%	0.4%
V. cardui	Ubx11a2+c5	50	5	3	3	2	4–4.5	500 : 500	10.0%	4.0%
		151	29	6	5	2	2–2.75	500:125:125	19.2%	1.3%
		361	18	13	16	6	0.5–2	500:125:125	5.0%	1.7%
	Total	562	52	22	24	10			9.3%	1.8%
V. cardui	Ubx11c5	168	99	27	26	3	3.75–4.75	250 : 125	58.9%	1.8%
		62	22	9	9	2	0.5–0.75	500 : 250	35.5%	3.2%
		131	93	8	8	3	1.5–3	500 : 250	71.0%	2.3%
		114	63	20	20	6	3.5–4.5	500 : 250	55.3%	5.3%
	Total	475	277	64	63	14			58.3%	2.9%
V. cardui	Ubx11b9	32	18	6	5	1	1.25–2.25	500 : 250	56.3%	3.1%
		63	49	9	6	1	3.5–4.5	500 : 250	77.8%	1.6%
	Total	95	67	15	11	2			70.5%	2.1%
J. coenia	Ubx11b9	41	13	13	13	3	2.5–4	125 : 62.5	31.7%	7.3%
		48	21	14	14	1	2–3	250 : 125	43.8%	2.1%
	Total	89	34	27	27	4			38.2%	4.5%
*: upper estimate, includes 16 fifth instars larvae that were dissected for immunostainings, of which 7 were mutants (as evidenced by ectopic UbdA in forewings), and 3 dissected mutant pupae