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(dose)]

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: Pax7Cas9 mice were administrated with different doses of single AAV-sgMyoD (high: 5×1011 vg/mouse; middle; 1×1011 vg/mouse; low: 0.2×1011 vg/mouse) virus intramuscularly at P10. Pax7Cas9 mice administrated with a high (5×1011 vg/mouse) dose of AAV9 virus containing pAAV9-sgRNA backbone without any sgRNA insertion were used as control. Muscle stem cells were isolated 4 weeks after injection and sgMyoD 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）是介导肌肉发育与损伤诱导的肌肉再生的肌肉干细胞。然而，相关研究的推进速度在一定程度上受制于构建基因工程小鼠的技术瓶颈。尽管CRISPR-Cas9在多种细胞系及物种中的基因组编辑易用性已被诸多研究证实，但其在内源性SCs中的应用仍有待阐明。本研究构建了肌肉特异性表达Cas9的小鼠模型，并通过腺相关病毒9型（AAV9）介导的向导RNA（short guide RNAs，sgRNAs）递送，在新生期幼年SCs中实现了高效的体内基因组编辑。同时本研究发现，尽管成年静息SCs可被AAV9高效转导，但其对CRISPR/Cas9编辑的耐受性较差。为实现幼年SCs的体内编辑，本研究以靶向肌肉生理关键基因MyoD的sgRNAs为概念验证，实现了MyoD位点的高效编辑，进而导致SCs的积累及SCs分化缺陷，该表型与已报道的MyoD基因敲除小鼠一致。后续将该系统应用于SCs命运转换相关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的肌肉再生过程中的独特功能。此外，本研究还发现Myc通过调控三维染色质架构来调控SCs的激活过程。综上，本研究成功构建了一套适用于内源性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：于出生后第10天（P10）对Pax7Cas9小鼠肌肉注射不同剂量的单剂AAV-sgMyoD病毒（高剂量：5×10^11 vg/只；中剂量：1×10^11 vg/只；低剂量：0.2×10^11 vg/只）。以注射高剂量（5×10^11 vg/只）不含任何sgRNA插入的pAAV9-sgRNA骨架AAV9病毒的Pax7Cas9小鼠作为对照组。于注射后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sgMyoD靶向位点，并进行深度测序。