Cardiac-Specific Gene Editing via an AAV9-Tnnt2-SaCas9-miR122TS Vector
The adeno-associated virus serotype 9 (AAV9)-delivered gene expression driven by the cardiac troponin T (Tnnt2) promoter is broadly considered to be cardiac-specific. However, in cases where low AAV expression is sufficient to trigger a profound biological effect in CRISPR/Cas9 gene editing, the ect...
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Bio-protocol LLC
2025-03-01
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| Online Access: | https://bio-protocol.org/en/bpdetail?id=5235&type=0 |
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| author | Luzi Yang Congting Guo Yueshen Sun Yuxuan Guo |
| author_facet | Luzi Yang Congting Guo Yueshen Sun Yuxuan Guo |
| author_sort | Luzi Yang |
| collection | DOAJ |
| description | The adeno-associated virus serotype 9 (AAV9)-delivered gene expression driven by the cardiac troponin T (Tnnt2) promoter is broadly considered to be cardiac-specific. However, in cases where low AAV expression is sufficient to trigger a profound biological effect in CRISPR/Cas9 gene editing, the ectopic AAV9-Tnnt2 expression and gene editing in the liver becomes non-negligible. MicroRNA122 is a microRNA that is specifically expressed in the liver. The incorporation of the microRNA122 target sequence (miR122TS) into the 3' untranslated region (UTR) of the AAV transgene could reduce ectopic gene expression in the liver. Here, we provide a protocol for sgRNA design, plasmid construction, AAV packaging, and in vivo validation of a new AAV9-Tnnt2-SaCas9-miR122TS vector using publicly available materials and tools. The application of this new vector enables cardiac-specific gene editing while circumventing leakages in the liver. |
| format | Article |
| id | doaj-art-069bdf64e9ed4a82836b80f9c38e5ebe |
| institution | DOAJ |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-069bdf64e9ed4a82836b80f9c38e5ebe2025-08-20T03:15:31ZengBio-protocol LLCBio-Protocol2331-83252025-03-0115510.21769/BioProtoc.5235Cardiac-Specific Gene Editing via an AAV9-Tnnt2-SaCas9-miR122TS VectorLuzi Yang0Congting Guo1Yueshen Sun2Yuxuan Guo3School of Basic Medical Sciences, Institute of Cardiovascular Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University Health Science Center, Beijing, ChinaSchool of Basic Medical Sciences, Institute of Cardiovascular Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University Health Science Center, Beijing, ChinaState Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Peking Union Medical College, Beijing, ChinaSchool of Basic Medical Sciences, Institute of Cardiovascular Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University Health Science Center, Beijing, ChinaBeijing Key Laboratory of Cardiovascular Receptors Research, Beijing, ChinaThe adeno-associated virus serotype 9 (AAV9)-delivered gene expression driven by the cardiac troponin T (Tnnt2) promoter is broadly considered to be cardiac-specific. However, in cases where low AAV expression is sufficient to trigger a profound biological effect in CRISPR/Cas9 gene editing, the ectopic AAV9-Tnnt2 expression and gene editing in the liver becomes non-negligible. MicroRNA122 is a microRNA that is specifically expressed in the liver. The incorporation of the microRNA122 target sequence (miR122TS) into the 3' untranslated region (UTR) of the AAV transgene could reduce ectopic gene expression in the liver. Here, we provide a protocol for sgRNA design, plasmid construction, AAV packaging, and in vivo validation of a new AAV9-Tnnt2-SaCas9-miR122TS vector using publicly available materials and tools. The application of this new vector enables cardiac-specific gene editing while circumventing leakages in the liver.https://bio-protocol.org/en/bpdetail?id=5235&type=0 |
| spellingShingle | Luzi Yang Congting Guo Yueshen Sun Yuxuan Guo Cardiac-Specific Gene Editing via an AAV9-Tnnt2-SaCas9-miR122TS Vector Bio-Protocol |
| title | Cardiac-Specific Gene Editing via an AAV9-Tnnt2-SaCas9-miR122TS Vector |
| title_full | Cardiac-Specific Gene Editing via an AAV9-Tnnt2-SaCas9-miR122TS Vector |
| title_fullStr | Cardiac-Specific Gene Editing via an AAV9-Tnnt2-SaCas9-miR122TS Vector |
| title_full_unstemmed | Cardiac-Specific Gene Editing via an AAV9-Tnnt2-SaCas9-miR122TS Vector |
| title_short | Cardiac-Specific Gene Editing via an AAV9-Tnnt2-SaCas9-miR122TS Vector |
| title_sort | cardiac specific gene editing via an aav9 tnnt2 sacas9 mir122ts vector |
| url | https://bio-protocol.org/en/bpdetail?id=5235&type=0 |
| work_keys_str_mv | AT luziyang cardiacspecificgeneeditingviaanaav9tnnt2sacas9mir122tsvector AT congtingguo cardiacspecificgeneeditingviaanaav9tnnt2sacas9mir122tsvector AT yueshensun cardiacspecificgeneeditingviaanaav9tnnt2sacas9mir122tsvector AT yuxuanguo cardiacspecificgeneeditingviaanaav9tnnt2sacas9mir122tsvector |