CRISPR/Cas9/AAV9-sgRNA Mediated In Vivo Genome Editing Reveals the Indispensability of Myc During Muscle Stem Cells Activation by Remodeling the 3D Chromatin [RNA-seq (myc&bcl6)]

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. Overall design: RNA-seq was performed in cultured muscle stem cells isoalted from Pax7cas9 mice infected with AAV9-dual sgMyc/sgBcl6 virus.

骨骼肌卫星细胞（skeletal muscle satellite cells, SCs）是负责肌肉发育及损伤诱导肌肉再生的肌肉干细胞。然而，SCs相关研究的进展在一定程度上受到基因修饰小鼠构建技术的限制。尽管已有诸多研究证实成簇规律间隔短回文重复序列相关蛋白9系统（CRISPR-Cas9）可在多种细胞系及不同物种中便捷地进行基因组编辑，但该技术在内源性SCs中的应用仍不甚明晰。本研究中，我们构建了肌肉特异性表达Cas9的小鼠，并通过腺相关病毒9型（adeno-associated virus serotype 9, AAV9）介导的单向导RNA（short guide RNAs, sgRNAs）递送，在出生后幼年阶段的SCs中实现了高效的体内基因组编辑。同时我们发现，尽管成年静息SCs可被AAV9高效转导，但它们难以被CRISPR-Cas9系统编辑。为实现体内幼年SCs的编辑，我们以肌分化因子1（myogenic differentiation 1, MyoD）——肌肉生理功能的关键调控基因——为靶点递送sgRNAs，作为概念验证研究。结果显示，MyoD基因座可被高效编辑，进而导致SCs的累积以及SCs分化缺陷，该表型与已报道的MyoD敲除小鼠表型一致。我们将该系统进一步应用于参与SCs命运转变的潜在关键转录因子（transcription factors, TFs）——Myc、Bcl6及Pknox2，揭示了它们在SCs激活早期及损伤诱导肌肉再生过程中的独特功能。此外，我们发现Myc通过调控三维染色质构象，协调SCs的激活过程。综上，我们在内源性SCs中建立了一套肌肉特异性的CRISPR-Cas9基因编辑平台，并阐明了调控SCs活性的关键因子的功能。整体实验设计：对从感染了双靶向sgMyc/sgBcl6的AAV9病毒的Pax7-Cas9小鼠中分离得到的体外培养肌肉干细胞进行RNA测序（RNA-seq）。