Table 2. CRISPR/Cas9-mediated gene editing for 10 genes delivered by microinjection and electroporation.

	Microinjection (NOD/ShiLtJ)				Electroporation (B6D2F2/J)
Target gene	Embryos transferred	Mice born	Mutant mice	Mutant percentage (%)	Embryos transferred	Mice born	Mutant mice	Mutant percentage (%)
Cd69	59	23	0	0	29	16	0	0
Cd226	61	19	4	21	20	13	0	0
Clec16a	57	0	NA	NA	25	16	2	13
Cyp27b1	64	23	12	52	28	20	0	0
Fut2	64	25	7	28	29	25	9	36
Ormdl3	62	19	17	89	26	15	2	13
Rgs1	62	18	8	44	28	19	5	26
Tlr7	66	22	6	27	30	15	0	0
Tlr8	60	15	1	7	29	22	3	14
Tnfsf9	61	21	15	71	25	14	0	0
Total	616	185		Live birth rate 30%	296	175		Live birth rate 59%

Zygotes were derived from B6D2F2/J and treated with acidic Tyrode’s solution for 10 sec before electroporation. A total of 30 embryos for each target gene were electroporated in a cuvette of 1-mm gap size and in 20 μl of TE/Opti-MEM at 1:1 volume ratio. Following electroporation, the embryos were transferred to 100 μl of M2 media and transferred into pseudopregnent female mice. For each live born mouse, PCR product at the target site was amplified from genomic DNA isolated from tail biopsies and analyzed by Sanger sequencing. Guide sequences for the 10 genes were chosen from the CRISPR Design software (crispr.mit.edu), targeting in-frame “ATG” downstream from the starting “ATG” if possible, with varied scores ranging from “41” for Cd69 to “90” for Fut2. NA, not applicable.