. 2024 Aug 30;70(6):356–361. doi: 10.1262/jrd.2024-054

Table 2. Development and mutation of zygotes cultured in different culture plates after simultaneous growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1) double-targeted lipofection.

Culture plate	No. of zygotes	No. (%) of zygotes developed to blastocysts	No. of blastocysts examined	No. (%) of GHR-edited blastocysts *			No. (%) of GGTA1-edited blastocysts *
Culture plate	No. of zygotes	No. (%) of zygotes developed to blastocysts	No. of blastocysts examined	WT	Mosaic	Average efficiency	WT	Mosaic	Average efficiency
4-well	200	33 (16.5 ± 4.6) ^a	18	18 (100)	0 (0.0)	–	16 (88.9) ^a	2 (11.1) ^a	16.3
25-well	197	64 (32.6 ± 2.3) ^b	20	20 (100)	0 (0.0)	–	11 (55.0) ^b	9 (45.0) ^b	9.8

Zona pellucida-free zygotes collected 10 h after in vitro fertilization were co-incubated with two guide RNAs (gRNAs) targeting GHR, GGTA1, and Lipofectamine 2000 (LP2000) for 5 h and cultured in 4-well or 25-well plates. Four replicates were performed. * Percentages were calculated by dividing the number of mutant blastocysts by the number of blastocysts examined. Average efficiency indicates the proportion of indel mutation events in mutant blastocysts, as determined using the Tracking of Indels by Decomposition (TIDE) analysis. ^a,bValues with different superscript letters are significantly different (P <0.05).