Screening of cis-acting elements for maintaining pluripotency of human embryonic stem cells
Objective To establish an inducible CRISPR library screening method for human embryonic stem cells (hESCs) to screen the cis-acting elements involed in the maintainance the pluripotency of hESCs. Methods The inducible Cas9 expression elements were inserted into the hESC genome to construct an indu...
| Main Author: | |
|---|---|
| Format: | Article |
| Language: | zho |
| Published: |
Institute of Basic Medical Sciences and Peking Union Medical College Hospital, Chinese Academy of Medical Sciences / Peking Union Medical College.
2021-05-01
|
| Series: | Jichu yixue yu linchuang |
| Subjects: | |
| Online Access: | http://journal11.magtechjournal.com/Jwk_jcyxylc/fileup/1001-6325/PDF/a210082.pdf |
| _version_ | 1797365814545874944 |
|---|---|
| author | SUN Meng-yao, LIU Si-qi, ZHOU Fan-qi, MA Yan-ni, YU Jia |
| author_facet | SUN Meng-yao, LIU Si-qi, ZHOU Fan-qi, MA Yan-ni, YU Jia |
| author_sort | SUN Meng-yao, LIU Si-qi, ZHOU Fan-qi, MA Yan-ni, YU Jia |
| collection | DOAJ |
| description | Objective To establish an inducible CRISPR library screening method for human embryonic stem cells (hESCs) to screen the cis-acting elements involed in the maintainance the pluripotency of hESCs. Methods The inducible Cas9 expression elements were inserted into the hESC genome to construct an inducible Cas9 stable expression strain (iCas9-hESC). Analyze the published chromatin interaction information in hESCs to identify the cis-acting elements that may be involved in the regulation of pluripotency genes, then to construct a double gRNA CRISPR knockout library. The CIRSPR library was used to infect iCas9-hESC at low multiplicity of infection followed by induction of Cas9 expression, and gene editing of the cells. Using the expression level of OCT4 as a standard to measure pluripotency, the cells after gene editing were stained with OCT4 and analyzed by flow cytometry to determine whether the knockout of cis-elements would affect the pluripotency of hESCs. Results The iCas9-hESC was successfully constructed, and the cells still maintained a pluripotent state. Construct a CRISPR knockout library of ciselements and successfully infect the target cells. Flow cytometric analysis of the expression level of OCT4 was performed on the cells after the library infection, and it was found that there were indeed some populations with decreased pluripotency in the cells after gene editing. Conclusions A feasible scheme for screening cis-acting elements involved in maintaining pluripotency in hESCs through CRISPR library is established. |
| first_indexed | 2024-03-08T16:54:17Z |
| format | Article |
| id | doaj.art-cb949fda924244a4b525a19bf2f72bc2 |
| institution | Directory Open Access Journal |
| issn | 1001-6325 |
| language | zho |
| last_indexed | 2024-03-08T16:54:17Z |
| publishDate | 2021-05-01 |
| publisher | Institute of Basic Medical Sciences and Peking Union Medical College Hospital, Chinese Academy of Medical Sciences / Peking Union Medical College. |
| record_format | Article |
| series | Jichu yixue yu linchuang |
| spelling | doaj.art-cb949fda924244a4b525a19bf2f72bc22024-01-05T03:05:08ZzhoInstitute of Basic Medical Sciences and Peking Union Medical College Hospital, Chinese Academy of Medical Sciences / Peking Union Medical College.Jichu yixue yu linchuang1001-63252021-05-01415636640Screening of cis-acting elements for maintaining pluripotency of human embryonic stem cellsSUN Meng-yao, LIU Si-qi, ZHOU Fan-qi, MA Yan-ni, YU Jia0State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC,Beijing 100005,ChinaObjective To establish an inducible CRISPR library screening method for human embryonic stem cells (hESCs) to screen the cis-acting elements involed in the maintainance the pluripotency of hESCs. Methods The inducible Cas9 expression elements were inserted into the hESC genome to construct an inducible Cas9 stable expression strain (iCas9-hESC). Analyze the published chromatin interaction information in hESCs to identify the cis-acting elements that may be involved in the regulation of pluripotency genes, then to construct a double gRNA CRISPR knockout library. The CIRSPR library was used to infect iCas9-hESC at low multiplicity of infection followed by induction of Cas9 expression, and gene editing of the cells. Using the expression level of OCT4 as a standard to measure pluripotency, the cells after gene editing were stained with OCT4 and analyzed by flow cytometry to determine whether the knockout of cis-elements would affect the pluripotency of hESCs. Results The iCas9-hESC was successfully constructed, and the cells still maintained a pluripotent state. Construct a CRISPR knockout library of ciselements and successfully infect the target cells. Flow cytometric analysis of the expression level of OCT4 was performed on the cells after the library infection, and it was found that there were indeed some populations with decreased pluripotency in the cells after gene editing. Conclusions A feasible scheme for screening cis-acting elements involved in maintaining pluripotency in hESCs through CRISPR library is established.http://journal11.magtechjournal.com/Jwk_jcyxylc/fileup/1001-6325/PDF/a210082.pdfhuman embryonic stem cell|pluripotency|cis-elements|crispr library |
| spellingShingle | SUN Meng-yao, LIU Si-qi, ZHOU Fan-qi, MA Yan-ni, YU Jia Screening of cis-acting elements for maintaining pluripotency of human embryonic stem cells Jichu yixue yu linchuang human embryonic stem cell|pluripotency|cis-elements|crispr library |
| title | Screening of cis-acting elements for maintaining pluripotency of human embryonic stem cells |
| title_full | Screening of cis-acting elements for maintaining pluripotency of human embryonic stem cells |
| title_fullStr | Screening of cis-acting elements for maintaining pluripotency of human embryonic stem cells |
| title_full_unstemmed | Screening of cis-acting elements for maintaining pluripotency of human embryonic stem cells |
| title_short | Screening of cis-acting elements for maintaining pluripotency of human embryonic stem cells |
| title_sort | screening of cis acting elements for maintaining pluripotency of human embryonic stem cells |
| topic | human embryonic stem cell|pluripotency|cis-elements|crispr library |
| url | http://journal11.magtechjournal.com/Jwk_jcyxylc/fileup/1001-6325/PDF/a210082.pdf |
| work_keys_str_mv | AT sunmengyaoliusiqizhoufanqimayanniyujia screeningofcisactingelementsformaintainingpluripotencyofhumanembryonicstemcells |