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| Status |
Public on Oct 27, 2021 |
| Title |
CRISPR/Cas9/AAV9-sgRNA Mediated In Vivo Genome Editing Reveals the Indispensability of Myc During Muscle Stem Cells Activation by Remodeling the 3D Chromatin [Hi-C] |
| Organism |
Mus musculus |
| Experiment type |
Other
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| Summary |
Skeletal muscle satellite cells (SCs) are muscle stem cells responsible for muscle development and injury induced muscle regeneration. The pace of SC related study, however, is constrained partially by the technological limitations in generating genetically modified mice. Although the ease of use of CRISPR-Cas9 in genome manipulation has been documented in many cell lines and various species, its application in endogenous SCs remains elusive. In this study, we generated muscle-specific Cas9-expressing mice and achieved robust in vivo genome editing in juvenile SCs at the postnatal stage through AAV9 mediated short guide RNAs (sgRNAs) delivery. We also found adult quiescent SCs are reluctant to CRISPR/Cas9 editing despite efficient AAV9 transduction. To edit juvenile SCs in vivo, as a proof-of-concept, we delivered sgRNAs targeting MyoD, a key gene critical for muscle physiology and showed an efficient editing at MyoD locus, resulting in accumulation of SCs and defects in SCs differentiation which resembled the phenotypes reported in MyoD knockout mice. Further application of this system on potential key transcription factors (TFs) involved in SC fate transition, Myc, Bcl6 and Pknox2, unveiled their distinct functions in the early stage of SC activation and injury induced muscle regeneration. In addition, we revealed that Myc orchestrated SCs activation through impinging on 3D chromatin architecture. Altogether we established a robust muscle restricted CRISPR/Cas9-based gene editing platform in endogenous SCs and elucidated the functionality of key factors governing SC activities.
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| Overall design |
The in situ Hi-C libraries were prepared as previously reported. Briefly, isolated SCs were digested using 100U DpnII overnight at 37 °C and then filled in with biotin for 1.5 hours at 37 °C; ligated for 4.5 hours at room temperature. DNA was purified by ethanol precipitation, and then sheared into 300-400bp fragments using Covaris S220. DNA fragments containing biotin were enriched by Dynabeads MyOne Streptavidin C1 (Invitrogen 65001) for 15min at room temperature, followed by end repairing, adaptor ligation and PCR amplification as described. At least two biological replicates were performed for both control and sgMyc groups. The libraries were then sequenced via the Illumina HiSeq X Ten system at Genewiz company.
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| Contributor(s) |
Huating W, Hao S, Liangqiang H, Yingzhe D, Yu Z |
| Citation(s) |
34534448 |
| Submission date |
Sep 13, 2020 |
| Last update date |
Oct 27, 2021 |
| Contact name |
Yingzhe Ding |
| E-mail(s) |
1155068845@link.cuhk.edu.hk
|
| Organization name |
The Chinese University of Hong Kong
|
| Department |
Chemical Pathology
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| Street address |
Rm503,Li Ka Shing Medical Science Build.,Prince of Wales Hosp.,30-32 Ngan SHing St.
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| City |
Hong Kong |
| State/province |
Shatin NT. |
| ZIP/Postal code |
999077 |
| Country |
Hong Kong |
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| Platforms (1) |
| GPL18480 |
Illumina HiSeq 1500 (Mus musculus) |
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| Samples (5)
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| This SubSeries is part of SuperSeries: |
| GSE134529 |
CRISPR/Cas9/AAV9-sgRNA Mediated In Vivo Genome Editing Reveals the Indispensability of Myc During Muscle Stem Cells Activation by Remodeling the 3D Chromatin |
|
| Relations |
| BioProject |
PRJNA663157 |
| SRA |
SRP282225 |
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