CRISPR/Cas9/AAV9-sgRNA Mediated In Vivo Genome Editing Reveals the Indispensability of Myc During Muscle Stem Cells Activation by Remodeling the 3D Chromatin [DEEP-seq(MyoD&Myc&Bcl6&Pknox2)]. CRISPR/Cas9/AAV9-sgRNA Mediated In Vivo Genome Editing Reveals the Indispensability of Myc During Muscle Stem Cells Activation by Remodeling the 3D Chromatin [DEEP-seq(MyoD&Myc&Bcl6&Pknox2)]

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: a middle dose (1×1011 vg/mouse) of single AAV9-dual sgMyoD, sgMyc,sgPknox2 or sgBcl6 virus was injected into Pax7Cas9 mice intramuscularly at P10.Muscle stem cells were isolated 4 weeks after injection and sgMyoD, sgMyc,sgPknox2 or sgBcl6 targeted locus was amplified from genomic DNA in freshly isolated satellite cells and subjected to deep sequencing.

骨骼肌卫星细胞（Skeletal muscle satellite cells, SCs）是负责肌肉发育与损伤诱导肌肉再生的肌肉干细胞。然而，相关研究的推进速度部分受制于构建基因修饰小鼠的技术瓶颈。尽管CRISPR-Cas9（成簇规律间隔短回文重复序列相关蛋白9）在多种细胞系及不同物种中用于基因组操作的便捷性已被广泛证实，但其在内源性SCs中的应用仍不甚明确。
本研究中，我们构建了肌肉特异性表达Cas9的小鼠，并通过AAV9（腺相关病毒9型）介导的sgRNA（短向导RNA，short guide RNAs）递送，在新生期幼年SCs中实现了高效的体内基因组编辑。同时我们发现，尽管AAV9可高效转导成年静息态SCs，但这类细胞对CRISPR/Cas9编辑的响应性较差。
为实现体内幼年SCs的编辑，我们以靶向肌肉生理关键基因MyoD的sgRNA开展概念验证实验，结果显示MyoD基因座可被高效编辑，进而引发SCs的积累及其分化缺陷，该表型与已报道的MyoD敲除小鼠表型一致。
我们进一步将该系统应用于参与SCs命运转换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，我们证实Myc通过调控三维染色质架构来介导SCs的激活。
综上，我们成功构建了一套针对内源性SCs的肌肉特异性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：向出生后第10天（P10）的Pax7Cas9小鼠肌肉内注射中等剂量（1×10^11 病毒基因组拷贝/只小鼠）的单AAV9载体，该载体携带双sgRNA（sgMyoD、sgMyc、sgPknox2或sgBcl6）。注射后4周分离肌肉干细胞，从新鲜分离的卫星细胞的基因组DNA中扩增sgRNA靶向的MyoD、Myc、sgPknox2或sgBcl6基因座，并进行深度测序。