Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system
CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GH...
Saved in:
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
The Society for Reproduction and Development
2024-08-01
|
| Series: | The Journal of Reproduction and Development |
| Subjects: | |
| Online Access: | https://www.jstage.jst.go.jp/article/jrd/70/6/70_2024-054/_pdf/-char/en |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850060540905259008 |
|---|---|
| author | Qingyi LIN Koki TAKEBAYASHI Nanaka TORIGOE Bin LIU Zhao NAMULA Maki HIRATA Fuminori TANIHARA Megumi NAGAHARA Takeshige OTOI |
| author_facet | Qingyi LIN Koki TAKEBAYASHI Nanaka TORIGOE Bin LIU Zhao NAMULA Maki HIRATA Fuminori TANIHARA Megumi NAGAHARA Takeshige OTOI |
| author_sort | Qingyi LIN |
| collection | DOAJ |
| description | CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneously double-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes. |
| format | Article |
| id | doaj-art-8e3940eab30f4a019a3a699e58a449bd |
| institution | DOAJ |
| issn | 0916-8818 1348-4400 |
| language | English |
| publishDate | 2024-08-01 |
| publisher | The Society for Reproduction and Development |
| record_format | Article |
| series | The Journal of Reproduction and Development |
| spelling | doaj-art-8e3940eab30f4a019a3a699e58a449bd2025-08-20T02:50:30ZengThe Society for Reproduction and DevelopmentThe Journal of Reproduction and Development0916-88181348-44002024-08-0170635636110.1262/jrd.2024-054jrdGenome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 systemQingyi LIN0Koki TAKEBAYASHI1Nanaka TORIGOE2Bin LIU3Zhao NAMULA4Maki HIRATA5Fuminori TANIHARA6Megumi NAGAHARA7Takeshige OTOI8Bio-Innovation Research Center, Tokushima University, Tokushima 779-3233, JapanBio-Innovation Research Center, Tokushima University, Tokushima 779-3233, JapanBio-Innovation Research Center, Tokushima University, Tokushima 779-3233, JapanBio-Innovation Research Center, Tokushima University, Tokushima 779-3233, JapanBio-Innovation Research Center, Tokushima University, Tokushima 779-3233, JapanBio-Innovation Research Center, Tokushima University, Tokushima 779-3233, JapanCenter for Development of Advanced Medical Technology, Jichi Medical University, Tochigi 329-0498, JapanBio-Innovation Research Center, Tokushima University, Tokushima 779-3233, JapanBio-Innovation Research Center, Tokushima University, Tokushima 779-3233, JapanCRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneously double-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.https://www.jstage.jst.go.jp/article/jrd/70/6/70_2024-054/_pdf/-char/encrispr/cas9 systemgrowth hormone receptor (ghr)glycoprotein alpha-galactosyltransferase 1 (ggta1)lipofectionporcine zygote |
| spellingShingle | Qingyi LIN Koki TAKEBAYASHI Nanaka TORIGOE Bin LIU Zhao NAMULA Maki HIRATA Fuminori TANIHARA Megumi NAGAHARA Takeshige OTOI Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system The Journal of Reproduction and Development crispr/cas9 system growth hormone receptor (ghr) glycoprotein alpha-galactosyltransferase 1 (ggta1) lipofection porcine zygote |
| title | Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system |
| title_full | Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system |
| title_fullStr | Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system |
| title_full_unstemmed | Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system |
| title_short | Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system |
| title_sort | genome editing of porcine zygotes via lipofection of two guide rnas using a crispr cas9 system |
| topic | crispr/cas9 system growth hormone receptor (ghr) glycoprotein alpha-galactosyltransferase 1 (ggta1) lipofection porcine zygote |
| url | https://www.jstage.jst.go.jp/article/jrd/70/6/70_2024-054/_pdf/-char/en |
| work_keys_str_mv | AT qingyilin genomeeditingofporcinezygotesvialipofectionoftwoguidernasusingacrisprcas9system AT kokitakebayashi genomeeditingofporcinezygotesvialipofectionoftwoguidernasusingacrisprcas9system AT nanakatorigoe genomeeditingofporcinezygotesvialipofectionoftwoguidernasusingacrisprcas9system AT binliu genomeeditingofporcinezygotesvialipofectionoftwoguidernasusingacrisprcas9system AT zhaonamula genomeeditingofporcinezygotesvialipofectionoftwoguidernasusingacrisprcas9system AT makihirata genomeeditingofporcinezygotesvialipofectionoftwoguidernasusingacrisprcas9system AT fuminoritanihara genomeeditingofporcinezygotesvialipofectionoftwoguidernasusingacrisprcas9system AT meguminagahara genomeeditingofporcinezygotesvialipofectionoftwoguidernasusingacrisprcas9system AT takeshigeotoi genomeeditingofporcinezygotesvialipofectionoftwoguidernasusingacrisprcas9system |